Therapeutic compositions including frataxin, lactoferrin, and mitochondrial energy generating enzymes, and uses thereof

ABSTRACT

Disclosed herein are methods and compositions for the treatment and/or prevention of diseases or conditions comprising administration of a therapeutic biological molecule, and/or naturally or artificially occurring derivatives, analogues, or pharmaceutically acceptable salts thereof, alone or in combination with one or more active agents (e.g., an aromatic-cationic peptide). The present technology provides compositions related to aromatic-cationic peptides linked to a therapeutic biological molecule and uses of the same. In some embodiments, the aromatic-cationic peptide comprises 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe-Lys-NH 2 , or D-Arg-2′,6′-Dmt-Lys-Phe-NH 2 .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/313,767, filed Nov. 23, 2016, which is the U.S. National Stage ofInternational Patent Application No. PCT/US2015/032728, filed May 27,2015, which claims the benefit of and priority to U.S. ProvisionalApplication No. 62/003,844, filed May 28, 2014, the contents of whichare incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

Disclosed herein are methods and compositions related to the treatmentand/or amelioration of diseases and conditions comprising administrationof a therapeutic biological molecule and/or naturally or artificiallyoccurring derivatives, analogues, or pharmaceutically acceptable saltsthereof, alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide). The present technology relates generallyto aromatic-cationic peptide compositions where the aromatic-cationicpeptide is conjugated to a therapeutic biological molecule and their usein the prevention and treatment of medical diseases and conditions.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art.

Biological cells are generally highly selective as to the molecules thatare allowed to pass through the cell membrane. As such, the delivery ofcompounds, such as small molecules and biological molecules into a cellis usually limited by the physical properties of the compound. The smallmolecules and biological molecules may, for example, be pharmaceuticallyactive compounds.

SUMMARY

The present technology provides compositions and methods useful in theprevention, treatment and/or amelioration of diseases and conditions.

A therapeutic biological molecule (TBM) includes those molecules foundin nature as well as synthesized biological molecules. TBMs include, butare not limited to polynucleotides, peptide nucleic acids, and polyaminoacids. In some embodiments, the polyamino acid sequence is a peptide,polypeptide, partial or full length protein, chimeric peptide sequence,chimeric polypeptide sequence or a chimeric protein sequence. In someembodiments, the polynucleotide sequence is double-stranded DNA,single-stranded DNA, antisense RNA, mRNA, siRNA, miRNA, a ribozyme, anRNA decoy, or an external guide sequence for ribozymes. TBMs useful incompositions of the present technology include, but are not limited to,e.g., frataxin, lactoferrin, or mitochondrial enzymes, such as, but notlimited to NADH-coenzyme Q oxidoreductase, succinate-Q oxidoreductase,electron transfer flavoprotein-Q oxidoreductase, Q-cytochrome coxidoreductase, cytochrome c oxidase, ATP synthase, pyruvatedehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase,α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinicdehydrogenase, fumarase, malate dehydrogenase, and pyruvate carboxylase.

In one aspect, the present disclosure provides a composition comprisinga therapeutic biological molecule (TBM), derivatives, analogues, orpharmaceutically acceptable salts thereof, alone or in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the composition further comprises one or moreadditional active agents such as cyclosporine, a cardiac drug, ananti-inflammatory, an anti-hypertensive drug, an antibody, an ophthalmicdrug, an antioxidant, a metal complexer, and an antihistamine.

In one aspect, the present disclosure provides a method for treating orpreventing mitochondrial permeability transition in a subject,comprising administering to the subject a therapeutically effectiveamount of a composition comprising TBMs, or derivatives, analogues, orpharmaceutically acceptable salts thereof, alone or in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method of treating adisease or condition characterized by mitochondrial permeabilitytransition, comprising administering a therapeutically effective amountof a composition comprising TBMs, or derivatives, analogues, orpharmaceutically acceptable salts thereof, alone or in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the disease or condition comprises a neurologicalor neurodegenerative disease or condition, ischemia, reperfusion,hypoxia, atherosclerosis, ureteral obstruction, diabetes, complicationsof diabetes, arthritis, liver damage, insulin resistance, diabeticnephropathy, acute renal injury, chronic renal injury, acute or chronicrenal injury due to exposure to nephrotoxic agents and/or radiocontrastdyes, hypertension, metabolic syndrome, an ophthalmic disease orcondition such as dry eye, diabetic retinopathy, cataracts, retinitispigmentosa, glaucoma, macular degeneration, choroidalneovascularization, retinal degeneration, oxygen-induced retinopathy,cardiomyopathy, ischemic heart disease, heart failure, hypertensivecardiomyopathy, vessel occlusion, vessel occlusion injury, myocardialinfarction, coronary artery disease, or oxidative damage.

In some embodiments, the neurological or neurodegenerative disease orcondition comprises Alzheimer's disease, Amyotrophic Lateral Sclerosis(ALS), Parkinson's disease, Huntington's disease or Multiple Sclerosis.

In some embodiments, the subject is suffering from ischemia or has ananatomic zone of no-reflow in one or more of cardiovascular tissue,skeletal muscle tissue, cerebral tissue and renal tissue.

In one aspect, the present disclosure provides a method for reducingCD36 expression in a subject in need thereof, comprising administeringto the subject an effective amount of a composition comprising TBMs, orderivatives, analogues, or pharmaceutically acceptable salts thereof,alone or in combination with one or more active agents. In someembodiments, the active agents include any one or more of thearomatic-cationic peptides shown in Section II. In some embodiments, thearomatic-cationic peptide is 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for treating orpreventing a disease or condition characterized by CD36 elevation in asubject in need thereof, comprising administering to the subject aneffective amount of a composition comprising TBMs, or derivatives,analogues, or pharmaceutically acceptable salts thereof, alone or incombination with one or more active agents. In some embodiments, theactive agents include any one or more of the aromatic-cationic peptidesshown in Section II. In some embodiments, the aromatic-cationic peptideis 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the subject is diagnosed as having, suspected ofhaving, or at risk of having atherosclerosis, inflammation, abnormalangiogenesis, abnormal lipid metabolism, abnormal removal of apoptoticcells, ischemia such as cerebral ischemia and myocardial ischemia,ischemia-reperfusion, ureteral obstruction, stroke, Alzheimer's Disease,diabetes, diabetic nephropathy, or obesity.

In one aspect, the present disclosure provides a method for reducingoxidative damage in a removed organ or tissue, comprising administeringto the removed organ or tissue an effective amount of a compositioncomprising TBMs, or derivatives, analogues, or pharmaceuticallyacceptable salts thereof, alone or in combination with one or moreactive agents. In some embodiments, the active agents include any one ormore of the aromatic-cationic peptides shown in Section II. In someembodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the removed organ comprises a heart, lung,pancreas, kidney, liver, or skin.

In one aspect, the present disclosure provides a method for preventingthe loss of dopamine-producing neurons in a subject in need thereof,comprising administering to the subject an effective amount of acomposition comprising TBMs, or derivatives, analogues, orpharmaceutically acceptable salts thereof, alone or in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the subject is diagnosed as having, suspected ofhaving, or at risk of having Parkinson's disease or ALS.

In one aspect, the present disclosure provides a method of reducingoxidative damage associated with a neurodegenerative disease in asubject in need thereof, comprising administering to the subject aneffective amount of a composition comprising TBMs, or derivatives,analogues, or pharmaceutically acceptable salts thereof, alone or incombination with one or more active agents. In some embodiments, theactive agents include any one or more of the aromatic-cationic peptidesshown in Section II. In some embodiments, the aromatic-cationic peptideis 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the neurodegenerative disease comprises Alzheimer'sdisease, Parkinson's disease, or ALS.

In one aspect, the present disclosure provides a method for preventingor treating a burn injury in a subject in need thereof, comprisingadministering to the subject an effective amount of a compositioncomprising TBMs, or derivatives, analogues, or pharmaceuticallyacceptable salts thereof, alone or in combination with one or moreactive agents. In some embodiments, the active agents include any one ormore of the aromatic-cationic peptides shown in Section II. In someembodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for treating orpreventing mechanical ventilation-induced diaphragm dysfunction in asubject in need thereof, comprising administering to the subject aneffective amount of a composition comprising TBMs, or derivatives,analogues, or pharmaceutically acceptable salts thereof, alone or incombination with one or more active agents. In some embodiments, theactive agents include any one or more of the aromatic-cationic peptidesshown in Section II. In some embodiments, the aromatic-cationic peptideis 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for treating orpreventing no reflow following ischemia-reperfusion injury in a subjectin need thereof, comprising administering to the subject an effectiveamount of a composition comprising TBMs, or derivatives, analogues, orpharmaceutically acceptable salts thereof, alone or in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for preventingnorepinephrine uptake in a subject in need of analgesia, comprisingadministering to the subject an effective amount of a compositioncomprising TBMs, or derivatives, analogues, or pharmaceuticallyacceptable salts thereof, alone or in combination with one or moreactive agents. In some embodiments, the active agents include any one ormore of the aromatic-cationic peptides shown in Section II. In someembodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for treating orpreventing drug-induced peripheral neuropathy or hyperalgesia in asubject in need thereof, comprising administering to the subject aneffective amount of a composition comprising TBMs, or derivatives,analogues, or pharmaceutically acceptable salts thereof, alone or incombination with one or more active agents. In some embodiments, theactive agents include any one or more of the aromatic-cationic peptidesshown in Section II. In some embodiments, the aromatic-cationic peptideis 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for inhibitingor suppressing pain in a subject in need thereof, comprisingadministering to the subject an effective amount of a compositioncomprising TBMs, or derivatives, analogues, or pharmaceuticallyacceptable salts thereof, alone or in combination with one or moreactive agents. In some embodiments, the active agents include any one ormore of the aromatic-cationic peptides shown in Section II. In someembodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one aspect, the present disclosure provides a method for treatingatherosclerotic renal vascular disease (ARVD) in a subject in needthereof, comprising administering to the subject an effective amount ofa composition comprising TBMs, or derivatives, analogues, orpharmaceutically acceptable salts thereof, alone or in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the composition comprises a TBM, derivative,analogue, or pharmaceutically acceptable salts thereof.

In some embodiments, the composition further comprises one or more of atleast one pharmaceutically acceptable pH-lowering agent; and at leastone absorption enhancer effective to promote bioavailability of theactive agent, and one or more lamination layers.

In some embodiments, the pH-lowering agent is selected from the groupconsisting of citric acid, tartaric acid and an acid salt of an aminoacid.

The present technology provides compositions comprising anaromatic-cationic peptide of the present technology conjugated to a TBMas well as methods for their use. Such molecules are referred tohereinafter as “peptide conjugates.” At least one TBM and at least onearomatic-cationic peptide associate to form a peptide conjugate. The TBMand aromatic-cationic peptide can associate by any method known to thosein the art. Suitable types of associations include chemical bonds andphysical bonds. Chemical bonds include, for example, covalent bonds andcoordinate bonds. Physical bonds include, for instance, hydrogen bonds,dipolar interactions, van der Waal forces, electrostatic interactions,hydrophobic interactions and aromatic stacking. In some embodiments, thepeptide conjugates have the general structure shown below:aromatic-cationic peptide-TBM

In some embodiments, the peptide conjugates have the general structureshown below:aromatic-cationic peptide-linker-TBM

The type of association between the TBM and aromatic-cationic peptidestypically depends on, for example, functional groups available on theTBM and functional groups available on the aromatic-cationic peptide.The peptide conjugate linker may be nonlabile or labile. The peptideconjugate linker may be enzymatically cleavable.

In one aspect, the present technology provides a peptide conjugatecomprising a TBM conjugated to an aromatic-cationic peptide, wherein thearomatic-cationic peptide is selected from the group consisting of:2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or any peptide described in Section II; andwherein the TBM is a compound described in Section I.

In some embodiments, the TBM is conjugated to the aromatic-cationicpeptide by a linker. In some embodiments, the TBM and aromatic-cationicpeptide are chemically bonded. In some embodiments, the TBM andaromatic-cationic peptide are physically bonded.

In some embodiments, the aromatic-cationic peptide and the TBM arelinked using a labile linkage that is hydrolyzed in vivo to uncouple thearomatic-cationic peptide and the TBM. In some embodiments, the labilelinkage comprises an ester linkage.

In another aspect, the present technology provides methods fordelivering an aromatic-cationic peptide and/or TBM to a cell, the methodcomprising contacting the cell with a peptide conjugate, wherein thepeptide conjugate comprises the TBM conjugated to an aromatic-cationicpeptide, wherein the aromatic-cationic peptide is selected from thegroup consisting of: 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or any peptidedescribed in Section II; and wherein the TBM is a compound described inSection I.

In some embodiments, the TBM is conjugated to the aromatic-cationicpeptide by a linker. In some embodiments, the TBM and aromatic-cationicpeptide are chemically bonded. In some embodiments, the TBM andaromatic-cationic peptide are physically bonded. In some embodiments,the aromatic-cationic peptide and the TBM are linked using a labilelinkage that is hydrolyzed in vivo to uncouple the aromatic-cationicpeptide and the TBM. In some embodiments, the labile linkage comprisesan ester linkage.

In another aspect, the present technology provides methods for treating,ameliorating or preventing a medical disease or condition in a subjectin need thereof, comprising administering a therapeutically effectiveamount of a composition comprising an aromatic-cationic peptide of thepresent technology conjugated to a TBM to the subject thereby treating,amelioration or preventing the medical disease or condition.

In some embodiments, the medical disease or condition is characterizedby mitochondrial permeability transition.

In some embodiments, the medical disease or condition comprises aneurological or neurodegenerative disease or condition, ischemia,reperfusion, hypoxia, atherosclerosis, ureteral obstruction, diabetes,complications of diabetes, arthritis, liver damage, insulin resistance,diabetic nephropathy, acute renal injury, chronic renal injury, acute orchronic renal injury due to exposure to nephrotoxic agents and/orradiocontrast dyes, hypertension, Metabolic Syndrome, an ophthalmicdisease or condition such as dry eye, diabetic retinopathy, cataracts,retinitis pigmentosa, glaucoma, macular degeneration, choroidalneovascularization, retinal degeneration, oxygen-induced retinopathy,cardiomyopathy, ischemic heart disease, heart failure, hypertensivecardiomyopathy, vessel occlusion, vessel occlusion injury, myocardialinfarction, coronary artery disease, oxidative damage. In someembodiments, the neurological or neurodegenerative disease or conditioncomprises Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Parkinson's disease, Huntington's disease or Multiple Sclerosis.

In some embodiments, the subject is suffering from ischemia or has ananatomic zone of no-reflow in one or more of cardiovascular tissue,skeletal muscle tissue, cerebral tissue and renal tissue.

In another aspect, the present technology provides methods for reducingCD36 expression in a subject in need thereof, comprising administeringto the subject an effective amount of a composition comprising anaromatic-cationic peptide of the present technology conjugated to a TBM.

In another aspect, the present technology provides methods for treating,ameliorating or preventing a medical disease or condition characterizedby CD36 elevation in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a compositioncomprising an aromatic-cationic peptide of the present technologyconjugated to a TBM.

In some embodiments, the subject is diagnosed as having, is suspected ofhaving, or at risk of having atherosclerosis, inflammation, abnormalangiogenesis, abnormal lipid metabolism, abnormal removal of apoptoticcells, ischemia such as cerebral ischemia and myocardial ischemia,ischemia-reperfusion, ureteral obstruction, stroke, Alzheimer's disease,diabetes, diabetic nephropathy, or obesity.

In another aspect, the present technology provides methods for reducingoxidative damage in a removed organ or tissue, comprising administeringto the removed organ or tissue a therapeutically effective amount of acomposition comprising an aromatic-cationic peptide of the presenttechnology conjugated to a TBM. In some embodiments, the removed organcomprises a heart, lung, pancreas, kidney, liver, or skin.

In another aspect, the present technology provides methods forpreventing the loss of dopamine-producing neurons in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a composition comprising an aromatic-cationicpeptide of the present technology conjugated to a TBM. In someembodiments, the subject is diagnosed as having, suspected of having, orat risk of having Parkinson's disease or ALS.

In another aspect, the present technology provides methods for reducingoxidative damage associated with a neurodegenerative disease in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a composition comprising anaromatic-cationic peptide of the present technology conjugated to a TBM.In some embodiments, the neurodegenerative diseases comprise Alzheimer'sdisease, Parkinson's disease, or ALS.

In another aspect, the present technology provides methods forpreventing or treating a burn injury in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a composition comprising an aromatic-cationic peptide of thepresent technology conjugated to a TBM.

In another aspect, the present technology provides methods for treatingor preventing mechanical ventilation-induced diaphragm dysfunction in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a composition comprising anaromatic-cationic peptide of the present technology conjugated to a TBM.

In another aspect, the present technology provides methods for treatingor preventing no reflow following ischemia-reperfusion injury in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a composition comprising anaromatic-cationic peptide of the present technology conjugated to a TBM.

In another aspect, the present technology provides methods forpreventing norepinephrine uptake in a subject in need of analgesia,comprising administering to the subject a therapeutically effectiveamount of a composition comprising an aromatic-cationic peptide of thepresent technology conjugated to a TBM.

In another aspect, the present technology provides methods for treating,ameliorating or preventing drug-induced peripheral neuropathy orhyperalgesia in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of a compositioncomprising an aromatic-cationic peptide of the present technologyconjugated to a TBM.

In another aspect, the present technology provides methods forinhibiting or suppressing pain in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising an aromatic-cationic peptide of the presenttechnology conjugated to a TBM.

In another aspect, the present technology provides methods for treatingatherosclerotic renal vascular disease (ARVD) in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a composition comprising an aromatic-cationicpeptide of the present technology conjugated to a TBM.

In some embodiments, the aromatic-cationic peptide is defined by FormulaA.

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In some embodiments, the peptide is defined by Formula B:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In some embodiments, the aromatic-cationic peptides of the presenttechnology have a core structural motif of alternating aromatic andcationic amino acids. For example, the peptide may be a tetrapeptidedefined by any of Formulas C to F set forth below:Aromatic-Cationic-Aromatic-Cationic  (Formula C)Cationic-Aromatic-Cationic-Aromatic  (Formula D)Aromatic-Aromatic-Cationic-Cationic  (Formula E)Cationic-Cationic-Aromatic-Aromatic  (Formula F)wherein, Aromatic is a residue selected from the group consisting of:Phe (F), Tyr (Y), and Trp (W). In some embodiments, the Aromatic residuemay be substituted with cyclohexylalanine (Cha). In some embodiments,the Cationic residue is a residue selected from the group consisting of:Arg (R), Lys (K), and His (H). In some embodiments, the Cationic residuemay be substituted with norleucine (Nle) or 2-amino-heptanoic acid(Ahe).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustrative example of an aromatic-cationic peptide ofthe present disclosure linked by a labile bond to a TBM.

FIG. 2 shows illustrative examples of aromatic-cationic peptides of thepresent disclosure linked by covalent attachment to self-immolatingmoieties.

FIGS. 3A, B, and C show illustrative examples of aromatic-cationicpeptides of the present disclosure incorporating spacer units to linkthe additional moieties to the peptide.

FIG. 4 shows illustrative peptide chemistry to form amide bonds, wherethe R₂ free amine is 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and R₁ is selectedfrom a linker bearing the formula: -(linker)-COOH; or where linkerconsists of one or more carbon atoms. In some embodiments, the linkerconsists of two or more carbon atoms.

FIGS. 5A and 5B show exemplary linking chemistry of the presentdisclosure. In FIG. 5A, R is a TBM containing a pendant COOH group andR′ is a linker bearing the formula: -(linker)-OH where linker consistsof at least one or more carbon atoms. In FIG. 5B, R is a linker bearingthe formula: -(linker)-COOH where linker consists of at least one ormore carbon atoms; and R′ is a TBM containing a pendant OH group.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the present technology are described below invarious levels of detail in order to provide a substantial understandingof the present technology.

The present technology provides compositions comprising anaromatic-cationic peptide of the present technology conjugated to a TBM.Such molecules are referred to hereinafter as peptide conjugates.

At least one TBM as described in Section I and at least onearomatic-cationic peptide as described in Section II associate to form apeptide conjugate. The TBM and aromatic-cationic peptide can associateby any method known to those in the art. Suitable types of associationsinclude chemical bonds and physical bonds. Chemical bonds include, forexample, covalent bonds and coordinate bonds. Physical bonds include,for instance, hydrogen bonds, dipolar interactions, van der Waal forces,electrostatic interactions, hydrophobic interactions and aromaticstacking.

In some embodiments, the peptide conjugates have the general structureshown below:aromatic-cationic peptide-TBM

In some embodiments, the peptide conjugates have the general structureshown below:aromatic-cationic peptide-linker-TBM

The type of association between the TBM and aromatic-cationic peptidestypically depends on, for example, functional groups available on theTBM and functional groups available on the aromatic-cationic peptide.The peptide conjugate linker may be nonlabile or labile. The peptideconjugate linker may be enzymatically cleavable.

While the peptide conjugates described herein can occur and can be usedas the neutral (non-salt) peptide conjugate, the description is intendedto embrace all salts of the peptide conjugates described herein, as wellas methods of using such salts of the peptide conjugates. In oneembodiment, the salts of the peptide conjugates comprisepharmaceutically acceptable salts. Pharmaceutically acceptable salts arethose salts which can be administered as drugs or pharmaceuticals tohumans and/or animals and which, upon administration, retain at leastsome of the biological activity of the free compound (neutral compoundor non-salt compound). The desired salt of a basic peptide conjugate maybe prepared by methods known to those of skill in the art by treatingthe compound with an acid. Examples of inorganic acids include, but arenot limited to, hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, and phosphoric acid. Examples of organic acids include, butare not limited to, formic acid, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, sulfonic acids, and salicylic acid. Salts of basicpeptide conjugates with amino acids, such as aspartate salts andglutamate salts, can also be prepared. The desired salt of an acidicpeptide conjugate can be prepared by methods known to those of skill inthe art by treating the compound with a base. Examples of inorganicsalts of acid conjugates include, but are not limited to, alkali metaland alkaline earth salts, such as sodium salts, potassium salts,magnesium salts, and calcium salts; ammonium salts; and aluminum salts.Examples of organic salts of acid peptide conjugates include, but arenot limited to, procaine, dibenzylamine, N-ethylpiperidine,N,N′-dibenzylethylenediamine, and triethylamine salts. Salts of acidicpeptide conjugates with amino acids, such as lysine salts, can also beprepared. The present technology also includes all stereoisomers andgeometric isomers of the peptide conjugates, including diastereomers,enantiomers, and cis/trans (E/Z) isomers. The present technology alsoincludes mixtures of stereoisomers and/or geometric isomers in anyratio, including, but not limited to, racemic mixtures.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this present technologybelongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the term “about” encompasses the range of experimentalerror that may occur in a measurement and will be clear to the skilledartisan.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “alkenyl” refers to unsaturated aliphaticgroups including straight-chain (linear), branched-chain, cyclic groups,and combinations thereof, having the number of carbon atoms specified,or if no number is specified, having up to 12 carbon atoms, whichcontain at least one double bond (—C═C—). All double bonds may beindependently either (E) or (Z) geometry, as well as arbitrary mixturesthereof. Examples of alkenyl groups include, but are not limited to,—CH₂—CH═CH—CH₃; and —CH₂—CH₂— cyclohexenyl, where the ethyl group can beattached to the cyclohexenyl moiety at any available carbon valence.

As used herein, the term “alkoxy” refers to an alkyl, alkenyl, alkynyl,or hydrocarbon chain linked to an oxygen atom and having the number ofcarbon atoms specified, or if no number is specified, having up to 12carbon atoms. Examples of alkoxy groups include, but are not limited to,groups such as methoxy, ethoxy, propyloxy (propoxy) (either n-propoxy ori-propoxy), and butoxy (either n-butoxy, i-butoxy, sec-butoxy, ortert-butoxy). The groups listed in the preceding sentence are alkoxygroups; an exemplary alkoxy substituent is methoxy.

As used herein, the term “alkyl” refers to saturated aliphatic groupsincluding straight-chain, branched-chain, cyclic groups, andcombinations thereof, having the number of carbon atoms specified, or ifno number is specified, having up to 12 carbon atoms. “Straight-chainalkyl” or “linear alkyl” groups refer to alkyl groups that are neithercyclic nor branched, commonly designated as “n-alkyl” groups. Examplesof alkyl groups include, but are not limited to, groups such as methyl,ethyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl,t-butyl, pentyl, n-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and adamantyl. Cycloalkyl groups can consist of one ring, including, butnot limited to, groups such as cycloheptyl, or multiple fused rings,including, but not limited to, groups such as adamantyl or norbornyl. Insome embodiments, the subset of alkyl groups is C₁-C₅ alkyl, which isintended to embrace methyl (Me), ethyl (Et), propyl (Pr), n-propyl(nPr), isopropyl (iPr), butyl (Bu), n-butyl (nBu), isobutyl (iBu),sec-butyl (sBu), t-butyl (tBu), cyclopropyl (cyclPr), cyclobutyl(cyclBu), cyclopropyl-methyl (cyclPr-Me), methyl-cyclopropane(Me-cyclPr), pentyl, n-pentyl, isopentyl, neopentyl, sec-pentyl,t-pentyl, 1,2-dimethylpropyl, cyclopentyl, and any other alkyl groupcontaining between one and five carbon atoms, where the C₁-C₅ alkylgroups can be attached via any valence on the C₁-C₅ alkyl groups.

Note that “C₀ alkyl,” when it appears, is intended to mean either anon-existent group, or a hydrogen, which will be understood by thecontext in which it appears. When a C₀ alkyl group appears as theterminal group on a chain, as for example in —(C═O)—C₀ alkyl, it isintended as a hydrogen atom; thus, —(C═O)—C₀ alkyl is intended torepresent (C═O)—H (an aldehyde). When a C₀ alkyl group appears betweentwo other groups, as, for example, in —(C═O)—C₀ alkyl-C₀-C₁₀ aryl, it isintended to be a nonentity, thus —(C═O)—C₀ alkyl-C₀-C₁₀ aryl represents—(C═O)—C₆-C₁₀ aryl. “C₁-C₆ alkyl” is intended to embrace a saturatedlinear, branched, cyclic, or a combination thereof, hydrocarbon of 1 to6 carbon atoms. Examples of “C₁-C₆ alkyl” are methyl, ethyl, n-propyl,isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl,cyclobutyl, cyclopropyl-methyl, methyl-cyclopropyl, pentyl where thepoint of attachment of the pentyl group to the remainder of the moleculecan be at any location on the pentyl moiety, cyclopentyl, hexyl wherethe point of attachment of the hexyl group to the remainder of themolecule can be at any location on the hexyl moiety, and cyclohexyl.

As used herein, the term “alkynyl” refers to unsaturated aliphaticgroups including straight-chain (linear), branched-chain, cyclic groups,and combinations thereof, having the number of carbon atoms specified,or if no number is specified, having up to 12 carbon atoms, whichcontain at least one triple bond (—C═C—). “Hydrocarbon chain” or“hydrocarbyl” refers to any combination of straight-chain, branchedchain, or cyclic alkyl, alkenyl, or alkynyl groups, and any combinationthereof. “Substituted alkenyl” “substituted alkynyl,” and “substitutedhydrocarbon chain” or “substituted hydrocarbyl” refer to the respectivegroup substituted with one or more substituents, including, but notlimited to, groups such as halogen, alkoxy, acyloxy, amino, hydroxyl,mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy,carboxaldehyde, carboalkoxy and carboxamide, or a functionality that canbe suitably blocked, if necessary for purposes of the presenttechnology, with a protecting group.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogues andamino acid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogues refer to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogues have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refer to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the terms “Aryl” or “Ar” refers to an aromatic cyclichydrocarbon group having a single ring (including, but not limited to,groups such as phenyl) or two or more condensed rings (including, butnot limited to, groups such as naphthyl or anthryl), and includes bothunsubstituted and substituted aryl groups. Aryls, unless otherwisespecified, contain from 6 to 12 carbon atoms in the ring portion. Arange for aryls is from 6 to 10 carbon atoms in the ring portion.“Substituted aryls” refers to aryls substituted with one or moresubstituents, including but not limited to, groups such as alkyl,alkenyl, alkynyl, hydrocarbon chains, halogen, alkoxy, acyloxy, amino,hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro,thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or afunctionality that can be suitably blocked, if necessary for purposes ofthe present technology, with a protecting group. “Aralkyl” designates analkyl-substituted aryl group, where any aryl can attach to the alkyl;the alkyl portion is a straight or branched chain of 1 to 6 carbonatoms. In some embodiments, the alkyl chain contains 1 to 3 carbonatoms. When an aralkyl group is indicated as a substituent, the aralkylgroup can be connected to the remainder of the molecule at any availablevalence on either its alkyl moiety or aryl moiety; e.g., the tolylaralkyl group can be connected to the remainder of the molecule byreplacing any of the five hydrogens on the aromatic ring moiety with theremainder of the molecule, or by replacing one of the alpha-hydrogens onthe methyl moiety with the remainder of the molecule. In someembodiments, the aralkyl group is connected to the remainder of themolecule via the alkyl moiety.

In some embodiments, the aryl group is phenyl, which can be substitutedor unsubstituted. Examples of substituents for substituted phenyl groupsinclude, but are not limited to, alkyl, halogen (chlorine (—Cl), bromine(—Br), iodine (—I), or fluorine (—F)), hydroxy (—OH), or alkoxy (such asmethoxy, ethoxy, n-propoxy or i-propoxy, n-butoxy, i-butoxy, sec-butoxy,or tert-butoxy). In some embodiments, substituted phenyl groups have oneor two substituents. In some embodiments, substituted phenyl groups haveone substituent.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in adisease or disorder or one or more signs or symptoms associated with adisease or disorder. In the context of therapeutic or prophylacticapplications, the amount of a composition administered to the subjectwill depend on the degree, type, and severity of the disease and on thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds. In the methods described herein, thetherapeutic compounds may be administered to a subject having one ormore signs or symptoms of a disease or disorder.

As used herein, the terms “halo” and “halogen” as used herein refer tothe Group VIIa elements (Group 17 elements in the 1990 IUPAC PeriodicTable, IUPAC Nomenclature of Inorganic Chemistry, Recommendations 1990)and include Cl, Br, F and I substituents. In some embodiments, halogensubstituents are Cl and F.

As used herein, the term “haloalkenyl” embraces any C₁-C₅ alkenylsubstituent having at least one halogen substituent; the halogen can beattached via any available valence on the C₁-C₅ alkenyl group. Onefurther subset of C₁-C₅ haloalkenyl is the subset with exactly onehalogen substituent. Another further subset of C₁-C₅ haloalkenyl is thesubset with exactly one chloro substituent. Another further subset ofC₁-C₅ haloalkenyl is the subset with exactly one fluoro substituent.Another further subset of C₁-C₅ haloalkenyl is the subset of C₁-C₅perhaloalkenyl; that is, C₁-C₅ alkenyl with all available valencesreplaced by halogens. Another further subset of C₁-C₅ haloalkenyl is thesubset of C₁-C₅ perfluoroalkenyl; that is, C₁-C₅ alkenyl with allavailable valences replaced by fluorines. Another further subset ofC₁-C₅ haloalkenyl is the subset of C₁-C₅ perchloroalkenyl; that is,C₁-C₅ alkenyl with all available valences replaced by chlorines.

As used herein, the term “haloalkyl” embraces any alkyl substituenthaving at least one halogen substituent. “C₁-C₆ haloalkyl” is intendedto embrace any C₁-C₆ alkyl substituent having at least one halogensubstituent; the halogen can be attached via any valence on the C₁-C₆alkyl group. Some examples of C₁-C₆ haloalkyl is —CF₃, —CCl₃, CHF₂,—CHCl₂, —CHBr₂, —CH₂F, —CH₂Cl.

As used herein, the term “haloalkynyl” embraces any C₁-C₅ alkynylsubstituent having at least one halogen substituent; the halogen can beattached via any available valence on the C₁-C₅ alkynyl group. Onefurther subset of C₁-C₅ haloalkynyl is the subset with exactly onehalogen substituent. Another further subset of C₁-C₅ haloalkynyl is thesubset with exactly one chloro substituent. Another further subset ofC₁-C₅ haloalkynyl is the subset with exactly one fluoro substituent.Another further subset of C₁-C₅ haloalkynyl is the subset of C₁-C₅perhaloalkynyl; that is, C₁-C₅ alkynyl with all available valencesreplaced by halogens. Another further subset of C₁-C₅ haloalkynyl is thesubset of C₁-C₅ perfluoroalkynyl; that is, C₁-C₅ alkynyl with allavailable valences replaced by fluorines. Another further subset ofC₁-C₅ haloalkynyl is the subset of C₁-C₅ perchloroalkynyl; that is,C₁-C₅ alkynyl with all available valences replaced by chlorines.

As used herein, the terms “heteroalkyl,” “heteroalkenyl,” and“heteroalkynyl” refer to alkyl, alkenyl, and alkynyl groups,respectively, that contain the number of carbon atoms specified (or ifno number is specified, having up to 12 carbon atoms) which contain oneor more heteroatoms as part of the main, branched, or cyclic chains inthe group. Heteroatoms include, but are not limited to, N, S, O, and P.In some embodiments, the heteroatoms are N or O. Heteroalkyl,heteroalkenyl, and heteroalkynyl groups may be attached to the remainderof the molecule either at a heteroatom (if a valence is available) or ata carbon atom. Examples of heteroalkyl groups include, but are notlimited to, groups such as —O—CH₃, CH₂—O—CH₃, —CH₂—CH₂—O—CH₃,—S—CH₂—CH₂—CH₃, —CH₂—CH(CH₃)—S—CH₃, —CH₂—CH₂—NH—CH₂—CH₂—,1-ethyl-6-propylpiperidino, and morpholino. Examples of heteroalkenylgroups include, but are not limited to, groups such asCH═CH—NH—CH(CH₃)—CH₂—. “Heteroaryl” or “HetAr” refers to an aromaticgroup having a single ring (including, but not limited to, examples suchas pyridyl, imidazolyl, thiophene, or furyl) or two or more condensedrings (including, but not limited to, examples such as indolizinyl orbenzothienyl) and having at least one hetero atom, including, but notlimited to, heteroatoms such as N, O, P, or S, within the ring. Examplesof heteroaryl include pyridine, pyrazine, imidazoline, thiazole,isothiazole, pyrazine, triazine, pyrimidine, pyridazine, pyrazole,thiophene, pyrrole, pyran, furan, indole, quinoline, quinazoline,benzimidazole, benzothiophene, benzofuran, benzoxazole, benzothiazole,benzotriazole, imidazo-pyridines, pyrazolo-pyridines, pyrazolo-pyrazine,acridine, carbazole, and the like. Unless otherwise specified,heteroalkyl, heteroalkenyl, heteroalkynyl, and heteroaryl groups havebetween one and five heteroatoms and between one and twelve carbonatoms. “Substituted heteroalkyl,” “substituted heteroalkenyl,”“substituted heteroalkynyl,” and “substituted heteroaryl” groups referto heteroalkyl, heteroalkenyl, heteroalkynyl, and heteroaryl groupssubstituted with one or more substituents, including, but not limitedto, groups such as alkyl, alkenyl, alkynyl, benzyl, hydrocarbon chains,halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy,phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxyand carboxamide, or a functionality that can be suitably blocked, ifnecessary for purposes of the present technology, with a protectinggroup. Examples of such substituted heteroalkyl groups include, but arenot limited to, piperazine, substituted at a nitrogen or carbon by aphenyl or benzyl group, and attached to the remainder of the molecule byany available valence on a carbon or nitrogen, —NH—SO₂-phenyl,NH—(C═O)O-alkyl, —NH—(C═O)O-alkylaryl, and —NH—(C═O)-alkyl. Ifchemically possible, the heteroatom(s) and/or the carbon atoms of thegroup can be substituted. The heteroatom(s) can also be in oxidizedform, if chemically possible.

When moieties, such as alkyl moieties, heteroaryl moieties, etc., areindicated as substituents, the substituent moiety can be attached to theremainder of the molecule at any point on the moiety where chemicallypossible (i.e., by using any available valence at a given point of themoiety, such as a valence made available by removing one or morehydrogen atoms from the moiety) unless otherwise specified. For example,in the moiety —(C═O)—C₀-C₈ alkyl-C₆-C₁₀ aryl-C₀-C₈ alkyl, if theleftmost C₀-C₈ alkyl group is a C₃ alkyl group, it can be attached tothe sp² carbon of the carbonyl group at any of the three carbon atoms inthe chain, unless otherwise specified. Likewise, the C₆-C₁₀ aryl groupcan be attached to the alkyl groups at any carbons in the aryl group,unless otherwise specified.

The terms “heterocycle”, “heterocyclic”, “heterocyclo”, and“heterocyclyl” is intended to encompass a monovalent, saturated, orpartially unsaturated, carbocyclic radical having one or more ringsincorporating one, two, three or four heteroatoms within the ring (e.g.nitrogen, oxygen, sulfur). Examples of heterocycles include morpholine,piperidine, piperazine, thiazolidine, pyrazolidine, pyrazoline,imidazolidine, pyrrolidine, tetrahydropyran, tetrahydrofuran,quinuclidine, and the like.

As used herein, an “isolated” or “purified” polypeptide or peptide issubstantially free of cellular material or other contaminatingpolypeptides from the cell or tissue source from which the agent isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. For example, an isolatedaromatic-cationic peptide would be free of materials that wouldinterfere with diagnostic or therapeutic uses of the agent. Suchinterfering materials may include enzymes, hormones and otherproteinaceous and nonproteinaceous solutes.

As used herein, the term “non-naturally-occurring” refers to acomposition which is not found in this form in nature. Anon-naturally-occurring composition can be derived from anaturally-occurring composition, e.g., as non-limiting examples, viapurification, isolation, concentration, chemical modification (e.g.,addition or removal of a chemical group), and/or, in the case ofmixtures, addition or removal of ingredients or compounds.Alternatively, a non-naturally-occurring composition can comprise or bederived from a non-naturally-occurring combination ofnaturally-occurring compositions. Thus, a non-naturally-occurringcomposition can comprise a mixture of purified, isolated, modifiedand/or concentrated naturally-occurring compositions, and/or cancomprise a mixture of naturally-occurring compositions in forms,concentrations, ratios and/or levels of purity not found in nature.

As used herein, the term “net charge” refers to the balance of thenumber of positive charges and the number of negative charges carried bythe amino acids present in the aromatic-cationic peptides of the presenttechnology. In this specification, it is understood that net charges aremeasured at physiological pH. The naturally occurring amino acids thatare positively charged at physiological pH include L-lysine, L-arginine,and L-histidine. The naturally occurring amino acids that are negativelycharged at physiological pH include L-aspartic acid and L-glutamic acid.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to one or more compounds that, in a statistical sample, reducesthe occurrence of the disorder or condition in the treated samplerelative to an untreated control sample, or delays the onset of one ormore symptoms of the disorder or condition relative to the untreatedcontrol sample.

As used herein, the term “protecting group” refers to a chemical groupthat exhibits the following characteristics: 1) reacts selectively withthe desired functionality in good yield to give a protected substratethat is stable to the projected reactions for which protection isdesired; 2) is selectively removable from the protected substrate toyield the desired functionality; and 3) is removable in good yield byreagents compatible with the other functional group(s) present orgenerated in such projected reactions. Examples of suitable protectinggroups can be found in Greene et al. (1991) Protective Groups in OrganicSynthesis, 3rd Ed. (John Wiley & Sons, Inc., New York). Amino protectinggroups include, but are not limited to, mesitylenesulfonyl (Mts),benzyloxycarbonyl (CBz or Z), t-butyloxycarbonyl (Boc),t-butyldimethylsilyl (TBS or TBDMS), 9-fluorenylmethyloxycarbonyl(Fmoc), tosyl, benzenesulfonyl, 2-pyridyl sulfonyl, or suitablephotolabile protecting groups such as 6-nitroveratryloxy carbonyl(Nvoc), nitropiperonyl, pyrenylmethoxycarbonyl, nitrobenzyl,α-,α-dimethyldimethoxybenzyloxycarbonyl (DDZ), 5-bromo-7-nitroindolinyl,and the like. Hydroxyl protecting groups include, but are not limitedto, Fmoc, TBS, photolabile protecting groups (such as nitroveratryloxymethyl ether (Nvom)), Mom (methoxy methyl ether), and Mem(methoxyethoxy methyl ether), NPEOC (4-nitrophenethyloxycarbonyl) andNPEOM (4-nitrophenethyloxymethyloxycarbonyl).

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the terms “subject,” “individual,” or “patient” can bean individual organism, a vertebrate, a mammal, or a human.

As used herein, a “synergistic therapeutic effect” refers to agreater-than-additive therapeutic effect which is produced by acombination of at least two agents, and which exceeds that which wouldotherwise result from the individual administration of agents. Forexample, lower doses of one or more agents may be used in treating adisease or disorder, resulting in increased therapeutic efficacy anddecreased side-effects.

As used herein, a “therapeutic biological molecule” (abbreviated as“TBM”) refers to those molecules found in nature as well as synthesizedbiological molecules. TBMs include, but are not limited topolynucleotides, peptide nucleic acids, and polyamino acids. In someembodiments, the polyamino acid sequence is a peptide, polypeptide,partial or full length protein, chimeric peptide sequence, chimericpolypeptide sequence or a chimeric protein sequence. In someembodiments, the polynucleotide sequence is double-stranded DNA,single-stranded DNA, antisense RNA, mRNA, siRNA, miRNA, a ribozyme, anRNA decoy, or an external guide sequence for ribozymes. TBMs useful incompositions of the present technology include, but are not limited to,e.g., frataxin, lactoferrin, or mitochondrial enzymes, such as, but notlimited to NADH-coenzyme Q oxidoreductase, succinate-Q oxidoreductase,electron transfer flavoprotein-Q oxidoreductase, Q-cytochrome coxidoreductase, cytochrome c oxidase, ATP synthase, pyruvatedehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase,α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinicdehydrogenase, fumarase, malate dehydrogenase, and pyruvate carboxylase.

As used herein, a “therapeutically effective amount” of a compoundrefers to compound levels in which the physiological effects of adisease or disorder are, at a minimum, ameliorated. A therapeuticallyeffective amount can be given in one or more administrations. The amountof a compound which constitutes a therapeutically effective amount willvary depending on the compound, the disorder and its severity, and thegeneral health, age, sex, body weight and tolerance to drugs of thesubject to be treated, but can be determined routinely by one ofordinary skill in the art.

“Treating” or “treatment” as used herein covers the treatment of adisease or disorder described herein, in a subject, such as a human, andincludes: (i) inhibiting a disease or disorder, i.e., arresting itsdevelopment; (ii) relieving a disease or disorder, i.e., causingregression of the disorder; (iii) slowing progression of the disorder;and/or (iv) inhibiting, relieving, or slowing progression of one or moresymptoms of the disease or disorder.

It is also to be appreciated that the various modes of treatment orprevention of medical diseases and conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved. The treatment may be a continuous prolongedtreatment for a chronic disease or a single, or few time administrationsfor the treatment of an acute condition.

I. Therapeutic Biological Molecules (TBMS)

TBMs described below are useful in compositions of the presenttechnology and include, but are not limited to, frataxin, lactoferrin,or mitochondrial enzymes, such as, but not limited to, NADH-coenzyme Qoxidoreductase, succinate-Q oxidoreductase, electron transferflavoprotein-Q oxidoreductase, Q-cytochrome c oxidoreductase, cytochromec oxidase, ATP synthase, pyruvate dehydrogenase, citrate synthase,aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase,succinyl-CoA synthetase, succinic dehydrogenase, fumarase, malatedehydrogenase, and pyruvate carboxylase.

Frataxin

Frataxin is a highly conserved iron binding protein. Human frataxin issynthesized as a 210 amino acid precursor that is imported to themitochondria via the mitochondrial targeting signal contained in theN-terminus. The frataxin precursor is subsequently cleaved to a mature14 kDa protein (residues 81-210).

Frataxin binds both Fe′ and Fe′ ions in an electrostatic manner andfunctions as an iron chaperone during Fe—S cluster assembly. Frataxindirectly binds to the central Fe—S cluster assembly complex, which iscomposed of Nfs1 enzyme and Isu scaffold protein. Nfs1 is a cysteinedesulfurase used in the synthesis of sulfur bioorganic derivatives andIsu is the transient scaffold protein on which the Fe—S clusterassembles. Frataxin increases the efficiency of Fe—S cluster formation,which is required to activate aconitase. Frataxin also plays a role inmitochondrial iron storage and heme biosynthesis by incorporatingmitochondrial iron into protoporphyrin (PIX).

Lactoferrin

Lactoferrin, also known as lactotransferrin, is a major iron-binding andmultifunctional protein of the transferrin family found in exocrinefluids such as breast milk, saliva, tears, and mucosal secretions.Lactoferrin is also present in secondary granules of neutrophils (PMNs).Lactoferrin can be purified from milk or recombinantly manufactured.Human lactoferrin is synthesized as a 710 amino acid precursor.Lactoferrins comprise two domains, each containing an iron-binding siteand an N-linked glycosylation site. Each domain can reversibly bind oneferric ion with high affinity. Lactoferrin also comprises an N-terminalbacteriocidal domain. Lactoferrins of the present technology alsocomprise lactoferrin derivatives, including allelic variants. Theprimary role of lactoferrin is to sequester free iron, thereby removingan essential substrate required for bacterial growth.

Electron Transport Chain Enzymes

Enzymes of the electron transport system use energy released from theoxidation of the reduced coenzyme NADH to pump protons across the innermembrane of the mitochondrion. This causes protons to build up in theintermembrane space, and generates an electrochemical gradient acrossthe membrane. The energy stored in this potential is then used by ATPsynthase to produce ATP.

NADH-coenzyme Q oxidoreductase, also known as NADH dehydrogenase orcomplex I, consists of 46 subunits and has a molecular mass of about1,000 kDa. The genes that encode the individual proteins are containedin both the cell nucleus and the mitochondrial genome. The reaction thatis catalyzed by this enzyme is the two electron oxidation of NADH bycoenzyme Q10 or ubiquinone in the mitochondrion membrane. The electronsenter complex I via flavin mononucleotide (FMN), a prosthetic groupattached to the complex. The addition of electrons to FMN converts it toits reduced form, FMNH₂. The electrons are then transferred through aseries of iron-sulfur clusters. As the electrons pass through thiscomplex, four protons are pumped from the matrix into the intermembranespace. Finally, the electrons are transferred from the chain ofiron-sulfur clusters to a ubiquinone molecule in the membrane. Reductionof coenzyme Q10 also contributes to the generation of a proton gradient,as two protons are taken up from the matrix as it is reduced toubiquinol (QH₂).

Defects in oxidative phosphorylation can be caused by mutations in genesencoding subunits of the electron transport chain. For example, thefunction of complex I subunits is altered by mutations in mitochondrialgenes including MTND1, MTND2, MTND4, and MIND6.

Succinate-Q oxidoreductase, also known as complex II or succinatedehydrogenase, is a second entry point to the electron transport chain.Complex II consists of four protein subunits and contains a bound flavinadenine dinucleotide (FAD) cofactor, iron-sulfur clusters, and a hemegroup. Complex II oxidizes succinate to fumarate and reduces ubiquinone.As this reaction releases less energy than the oxidation of NADH,complex II does not transport protons across the membrane and does notcontribute to the proton gradient. Complex II is the only enzyme thatparticipates in both the citric acid cycle and the electron transportchain.

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Qoxidoreductase), also known as electron transferring-flavoproteindehydrogenase, is a third entry point to the electron transport chain.It is an enzyme that accepts electrons from electron-transferringflavoprotein in the mitochondrial matrix, and uses these electrons toreduce ubiquinone. This enzyme contains a flavin and an iron-sulfurcluster that is attached to the surface of the membrane and does notcross the lipid bilayer.

Q-cytochrome c oxidoreductase is also known as cytochrome c reductase,cytochrome bc1 complex, or complex III. In mammals, this enzyme is adimer, with each subunit complex containing 11 protein subunits, aniron-sulfur cluster and three cytochromes: one cytochrome c1 and two bcytochromes. The iron atoms inside complex III's heme groups alternatebetween a reduced ferrous (+2) and oxidized ferric (+3) state as theelectrons are transferred through the protein.

The reaction catalyzed by complex III is the oxidation of one moleculeof ubiquinol and the reduction of two molecules of cytochrome c, whichcarries only one electron. As only one of the electrons can betransferred from the QH₂ donor to a cytochrome c acceptor at a time, thereaction mechanism of complex III occurs in two steps called the Qcycle. In the first step, the enzyme binds three substrates, first, QH₂,which is then oxidized, with one electron being passed to the secondsubstrate, cytochrome c. The two protons released from QH₂ pass into theintermembrane space. The third substrate is Q, which accepts the secondelectron from the QH₂ and is reduced to Q⁻, which is the ubisemiquinonefree radical. The first two substrates are released, but theubisemiquinone intermediate remains bound. In the second step, a secondmolecule of QH₂ is bound and again passes its first electron to acytochrome c acceptor. The second electron is passed to the boundubisemiquinone, reducing it to QH₂ as it gains two protons from themitochondrial matrix. This QH₂ is then released from the enzyme. Ascoenzyme Q is reduced to ubiquinol on the inner side of the membrane andoxidized to ubiquinone on the other, a net transfer of protons acrossthe membrane occurs, adding to the proton gradient.

Cytochrome c oxidase, also known as complex IV, is the final proteincomplex in the electron transport chain. The mammalian enzyme contains13 subunits, two heme groups, as well as multiple metal ion cofactors—inall, three atoms of copper, one of magnesium and one of zinc. Thisenzyme mediates the final reaction in the electron transport chain andtransfers electrons to oxygen, while pumping protons across themembrane. The reaction catalyzed is the oxidation of cytochrome c andthe reduction of oxygen. The final electron acceptor oxygen is reducedto water in this step. Both the direct pumping of protons and theconsumption of matrix protons in the reduction of oxygen contribute tothe proton gradient.

Defects in oxidative phosphorylation can be caused by mutations in genesencoding complex IV subunits as well as by mutations in genes involvedin complex IV assembly and in processes that affect complex IVbiogenesis. Mutations in mitochondrial genes MTCO1, MTCO2, and MTCO3,and in nuclear genes COX10, COX6B1, SCO1, and SCO2, are implicated incomplex IV deficiency. Mutations in nuclear genes SURF1 and COX15 arelinked to alterations in complex IV biogenesis.

ATP synthase, also called complex V, is the final enzyme in theoxidative phosphorylation pathway. The enzyme uses the energy stored ina proton gradient across a membrane to drive the synthesis of ATP fromADP and phosphate (Pi). Estimates of the number of protons required tosynthesize one ATP have ranged from three to four. This phosphorylationreaction is an equilibrium, which can be shifted by altering theproton-motive force. In the absence of a proton-motive force, the ATPsynthase reaction will run from right to left, hydrolyzing ATP andpumping protons out of the matrix across the membrane. However, when theproton-motive force is high, the reaction is forced to run in theopposite direction, allowing protons to flow down their concentrationgradient and turning ADP into ATP.

ATP synthase is a massive protein complex with a mushroom-like shape.The mammalian enzyme complex contains 16 subunits and has a mass ofapproximately 600 kDa. The portion embedded within the membrane iscalled F₀ and contains a ring of c subunits and the proton channel. Thestalk and the ball-shaped headpiece are collectively called Fi and isthe site of ATP synthesis. The ball-shaped complex at the end of the Fiportion contains six proteins of two different kinds (three α subunitsand three β subunits), whereas the stalk consists of one protein: the γsubunit, with the tip of the stalk extending into the ball of a and βsubunits. Both the α and β subunits bind nucleotides, but only the βsubunits catalyze the ATP synthesis reaction. As protons cross themembrane through the channel in the base of ATP synthase, the F₀proton-driven motor rotates. This rotating ring of c subunits in turndrives the rotation of the central axle (the γ subunit stalk) within theα and β subunits. This movement of the tip of the γ subunit within theball of α and β subunits provides the energy for the active sites in theβ subunits to undergo a cycle of movements that produces and thenreleases ATP.

This ATP synthesis reaction is called the binding change mechanism andinvolves the active site of a β subunit cycling between three states. Inthe “open” state, ADP and phosphate enter the active site. The proteinthen closes up around the molecules and binds them loosely—the “loose”state. The enzyme then changes shape again and forces these moleculestogether, with the active site in the resulting “tight” state bindingthe newly produced ATP molecule with very high affinity. Finally, theactive site cycles back to the open state, releasing ATP and bindingmore ADP and phosphate during the next cycle.

Defects in oxidative phosphorylation can be caused by mutations in genesencoding complex V subunits. Mutations in the mitochondrial gene MTATP6are linked to altered complex V subunits.

Citric Acid Cycle Enzymes

The citric acid cycle is a key component of the metabolic pathway bywhich all aerobic organisms generate energy. Through catabolism ofsugars, fats, and proteins, a two-carbon organic product acetate in theform of acetyl-CoA is produced. Acetyl-CoA along with two equivalents ofwater is consumed by the citric acid cycle producing two equivalents ofcarbon dioxide (CO₂) and one equivalent of Coenzyme A. In addition, onecomplete turn of the cycle converts three equivalents of nicotinamideadenine dinucleotide (NAD⁺) into three equivalents of reduced NADH, oneequivalent of ubiquinone (Q) into one equivalent of reduced ubiquinone(QH₂), and one equivalent each of guanosine diphosphate (GDP) andinorganic phosphate (P_(i)) into one equivalent of guanosinetriphosphate (GTP). The NADH and QH₂ generated by the citric acid cycleare in turn used by the oxidative phosphorylation pathway to generateenergy-rich adenosine triphosphate (ATP).

Citrate synthase exists in nearly all living cells and stands as apace-making enzyme in the first step of the Citric Acid Cycle. Citratesynthase is encoded by nuclear DNA, synthesized using cytoplasmicribosomes, then transported into the mitochondrial matrix. Citratesynthase catalyzes the condensation reaction of the two-carbon acetateresidue from acetyl coenzyme A and a molecule of four-carbonoxaloacetate to form the six-carbon citrate. Oxaloacetate will beregenerated after the completion of one round of the Krebs Cycle.Oxaloacetate is the first substrate to bind to the enzyme. This inducesthe enzyme to change its conformation, and creates a binding site forthe acetyl-CoA. Only when this citroyl-CoA has formed will anotherconformational change cause thioester hydrolysis and release coenzyme A.This ensures that the energy released from the thioester bond cleavagewill drive the condensation.

Aconitase is an enzyme that catalyzes the stereo-specific isomerizationof citrate to isocitrate via cis-aconitate in the citric acid cycle. Theiron-sulfur cluster of aconitase reacts directly with an enzymesubstrate. Aconitase has an active [Fe4S4]²⁺ cluster, which may convertto an inactive [Fe3S4]⁺ form. Three cysteine (Cys) residues have beenshown to be ligands of the [Fe4S4] center. In the inactive form, itsstructure is divided into four domains. Counting from the N-terminus,only the first three of these domains are involved in close interactionswith the [3Fe-4S] cluster, but the active site consists of residues fromall four domains, including the larger C-terminal domain. The Fe—Scluster and a SO₄ ²⁻ anion also reside in the active site. When theenzyme is activated, it gains an additional iron atom, creating a[4Fe-4S] cluster.

Isocitrate dehydrogenase (IDH) is an enzyme that catalyzes the oxidativedecarboxylation of isocitrate, producing alpha-ketoglutarate(α-ketoglutarate) and CO₂. This is a two-step process, which involvesoxidation of isocitrate to oxalosuccinate, followed by thedecarboxylation of the carboxyl group beta to the ketone, formingalpha-ketoglutarate. In humans, IDH exists in three isoforms: IDH3catalyzes the third step of the citric acid cycle while converting NAD+to NADH in the mitochondria.

The oxoglutarate dehydrogenase complex (OGDC) or α-ketoglutaratedehydrogenase complex is an enzyme complex that catalyzes the followingreaction:α-ketoglutarate+NAD⁺+CoA Succinyl CoA+CO₂+NADH.

This reaction proceeds in three steps: (1) decarboxylation ofα-ketoglutarate, (2) reduction of NAD+ to NADH, and (3) subsequenttransfer to CoA, which forms the end product, succinyl CoA. The energyneeded for this oxidation is conserved in the formation of a thioesterbond of succinyl CoA.

Succinyl coenzyme A synthetase is a mitochondrial enzyme that catalyzesthe reversible reaction of succinyl-CoA to succinate. The enzymefacilitates the coupling of this reaction to the formation of anucleoside triphosphate molecule (either GTP or ATP) from an inorganicphosphate molecule and a nucleoside diphosphate molecule (either GDP orADP). The reaction takes place by a three-step mechanism. The first stepinvolves displacement of CoA from succinyl CoA by a nucleophilicinorganic phosphate molecule to form succinyl phosphate. The enzyme thenutilizes a histidine residue to remove the phosphate group from succinylphosphate and generate succinate. Finally, the phosphorylated histidinetransfers the phosphate group to a nucleoside diphosphate, whichgenerates the high-energy carrying nucleoside triphosphate.

Succinate dehydrogenase or succinate-coenzyme Q reductase (SQR) orrespiratory Complex II is an enzyme complex bound to the innermitochondrial membrane. It is the only enzyme that participates in boththe citric acid cycle and the electron transport chain. SQR catalyzesthe oxidation of succinate to fumarate with the reduction of ubiquinoneto ubiquinol. This occurs in the inner mitochondria membrane by couplingthe two reactions together.

Mitochondrial SQRs are composed of four subunits: two hydrophilic andtwo hydrophobic. The first two subunits, a flavoprotein (SdhA) and aniron-sulfur protein (SdhB), are hydrophilic. SdhA contains a covalentlyattached flavin adenine dinucleotide (FAD) cofactor and the succinatebinding site and SdhB contains three iron-sulfur clusters: [2Fe-2S],[4Fe-4S], and [3Fe-4S]. The second two subunits are hydrophobic membraneanchor subunits, SdhC and SdhD. The subunits form a membrane-boundcytochrome b complex with six transmembrane helices containing one hemeb group and a ubiquinone-binding site. Two phospholipid molecules, onecardiolipin and one phosphatidylethanolamine, are also found in the SdhCand SdhD subunits and serve to occupy the hydrophobic space below theheme b.

Fumarase (or fumarate hydratase) is an enzyme that catalyzes thereversible hydration/dehydration of fumarate to malate. Fumarase comesin two forms: mitochondrial and cytosolic. The mitochondrial isoenzymeis involved in the Citric Acid Cycle, and the cytosolic isoenzyme isinvolved in the metabolism of amino acids and fumarate. The function offumarase in the citric acid cycle is to facilitate a transition step inthe production of energy in the form of NADH. The primary binding siteon fumarase is known as catalytic site A. Studies have revealed thatcatalytic site A is composed of amino acid residues from three of thefour subunits within the tetrameric enzyme. Two potential acid-basecatalytic residues in the reaction include His 188 and Lys 324.

Malate dehydrogenase (MDH) is an enzyme that reversibly catalyzes theoxidation of malate to oxaloacetate using the reduction of NAD+ to NADH.This reaction is part of many metabolic pathways, including the citricacid cycle. Pyruvate in the mitochondria is acted upon by pyruvatecarboxylase to form oxaloacetate, a citric acid cycle intermediate. Inorder to facilitate the transfer of oxaloacetate out of themitochondrion, malate dehydrogenase reduces oxaloacetate to malate, andit then traverses the inner mitochondrial membrane. Once in the cytosol,the malate is oxidized back to oxaloacetate by cytosolic malatedehydrogenase. The active site of malate dehydrogenase is a hydrophobiccavity within the protein complex that has specific binding sites forthe substrate and its coenzyme, NAD+. In its active state, MDH undergoesa conformational change that encloses the substrate to minimize solventexposure and to position key residues in closer proximity to thesubstrate. The three residues in particular that comprise a catalytictriad are histidine (His-195), aspartate (Asp-168), both of which worktogether as a proton transfer system, and arginines (Arg-102, Arg-109,Arg-171), which secure the substrate. Kinetic studies show that MDHenzymatic activity is ordered. NAD+/NADH is bound before the substrate.

Pyruvate dehydrogenase complex (PDC) is a complex of three enzymes thattransform pyruvate into acetyl-CoA by a process called pyruvatedecarboxylation. Acetyl-CoA may then be used in the citric acid cycle tocarry out cellular respiration, and this complex links the glycolysismetabolic pathway to the citric acid cycle, ultimately releasing energyvia NADH. Pyruvate decarboxylation is also known as the “pyruvatedehydrogenase reaction” because it also involves the oxidation ofpyruvate. Pyruvate dehydrogenase complex is located in the mitochondrialmatrix of eukaryotes. It is organized in dodecahedral symmetry, andconsists of a total of 96 subunits, organized into three functionalproteins: pyruvate dehydrogenase, dihydrolipoyl transacetylase, anddihydrolipoyl dehydrogenase.

Pyruvate carboxylase (PC) is an enzyme of the ligase class thatcatalyzes the carboxylation of pyruvate to form oxaloacetate. The enzymeis a mitochondrial protein containing a biotin prosthetic group,requiring magnesium or manganese and acetyl CoA. Most well characterizedforms of active PC consist of four identical subunits arranged in atetrahedron-like structure. Each subunit contains a single biotin moietyacting as a swinging arm to transport carbon dioxide to the catalyticsite that is formed at the interface between adjacent monomers. Eachsubunit of the functional tetramer contains four domains: the biotincarboxylation (BC) domain, the transcarboxylation (CT) domain, thebiotin carboxyl carrier (BCCP) domain and the PC tetramerization (PT)domain. Pyruvate carboxylase uses a covalently attached biotin cofactorwhich is used to catalyze the ATP-dependent carboxylation of pyruvate tooxaloacetate in two steps. Biotin is initially carboxylated at the BCactive site by ATP and bicarbonate. The carboxyl group is subsequentlytransferred by carboxybiotin to a second active site in the CT domain,where pyruvate is carboxylated to generate oxaloacetate. The BCCP domaintransfers the tethered cofactor between the two remote active sites.

II. Aromatic-Cationic Peptides as Active Agents

The aromatic-cationic peptides of the present technology arewater-soluble, highly polar, and can readily penetrate cell membranes.

The aromatic-cationic peptides of the present technology include aminimum of three amino acids, covalently joined by peptide bonds.

The maximum number of amino acids present in the aromatic-cationicpeptides of the present technology is about twenty amino acidscovalently joined by peptide bonds. In some embodiments, the maximumnumber of amino acids is about twelve. In some embodiments, the maximumnumber of amino acids is about nine. In some embodiments, the maximumnumber of amino acids is about six. In some embodiments, the maximumnumber of amino acids is four.

In some aspects, the present technology provides an aromatic-cationicpeptide or a pharmaceutically acceptable salt thereof such as acetatesalt or trifluoroacetate salt. In some embodiments, the peptidecomprises at least one net positive charge; a minimum of three aminoacids; a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1; and

a relationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1.

In some embodiments, the peptide comprises the amino acid sequence2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In some embodiments, the peptide comprisesone or more of the peptides of Table A:

TABLE A Tyr-D-Arg-Phe-Lys-NH₂ D-Arg-Dmt-Lys-Phe-NH₂D-Arg-Dmt-Phe-Lys-NH₂ D-Arg-Phe-Lys-Dmt-NH₂ D-Arg-Phe-Dmt-Lys-NH₂D-Arg-Lys-Dmt-Phe-NH₂ D-Arg-Lys-Phe-Dmt-NH₂ D-Arg-Dmt-Lys-Phe-Cys-NH₂D-Arg-Dmt-Lys-Phe-Glu-Cys-Gly-NH₂ D-Arg-Dmt-Lys-Phe-Ser-Cys-NH₂D-Arg-Dmt-Lys-Phe-Gly-Cys-NH₂ Phe-Lys-Dmt-D-Arg-NH₂Phe-Lys-D-Arg-Dmt-NH₂ Phe-D-Arg-Phe-Lys-NH₂ Phe-D-Arg-Phe-Lys-Cys-NH₂Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂ Phe-D-Arg-Phe-Lys-Ser-Cys-NH₂Phe-D-Arg-Phe-Lys-Gly-Cys-NH₂ Phe-D-Arg-Dmt-Lys-NH₂Phe-D-Arg-Dmt-Lys-Cys-NH₂ Phe-D-Arg-Dmt-Lys-Glu-Cys-Gly-NH₂Phe-D-Arg-Dmt-Lys-Ser-Cys-NH₂ Phe-D-Arg-Dmt-Lys-Gly-Cys-NH₂Phe-D-Arg-Lys-Dmt-NH₂ Phe-Dmt-D-Arg-Lys-NH₂ Phe-Dmt-Lys-D-Arg-NH₂Lys-Phe-D-Arg-Dmt-NH₂ Lys-Phe-Dmt-D-Arg-NH₂ Lys-Dmt-D-Arg-Phe-NH₂Lys-Dmt-Phe-D-Arg-NH₂ Lys-D-Arg-Phe-Dmt-NH₂ Lys-D-Arg-Dmt-Phe-NH₂D-Arg-Dmt-D-Arg-Phe-NH₂ D-Arg-Dmt-D-Arg-Dmt-NH₂ D-Arg-Dmt-D-Arg-Tyr-NH₂D-Arg-Dmt-D-Arg-Trp-NH₂ Trg-D-Arg-Tyr-Lys-NH₂ Trp-D-Arg-Trp-Lys-NH₂Trp-D-Arg-Dmt-Lys-NH₂ D-Arg-Trp-Lys-Phe-NH₂ D-Arg-Trp-Phe-Lys-NH₂D-Arg-Trp-Lys-Dmt-NH₂ D-Arg-Trp-Dmt-Lys-NH₂ D-Arg-Lys-Trp-Phe-NH₂D-Arg-Lys-Trp-Dmt-NH₂ Cha-D-Arg-Phe-Lys-NH₂ Ala-D-Arg-Phe-Lys-NH₂2′,6′-Dmp-D-Arg-2′,6′-Dmt-Lys-NH₂ 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂2′,6′-Dmt-D-Arg-Phe-Orn-NH₂2′,6′-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)-NH₂2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Cit-PheLys-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheArg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe- Tyr-D-Arg-GlyAsp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-Arg-2′,6′-Dmt-Lys-Phe-NH₂D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂ D-His-Glu-Lys-Tyr-D-Phe-ArgD-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp- NH₂D-Tyr-Trp-Lys-NH₂Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg- D-Met-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp. Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂ Lys-D-Arg-Tyr-NH₂Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂ Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Met-Tyr-D-Arg-Phe-Arg-NH₂ Met-Tyr-D-Lys-Phe-Arg Phe-Arg-D-His-AspPhe-D-Arg-2′,6′-Dmt-Lys-NH₂ Phe-D-Arg-HisPhe-D-Arg-Lys-Trp-Tyr-D-Arg-HisPhe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrThr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysTyr-D-Arg-Phe-Lys-Glu-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Tyr-His-D-Gly-MetVal-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-PhePhe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysGlu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr- Arg-D-Met-NH₂Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe- Tyr-D-Arg-GlyGly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp D-Arg-Tyr-Lys-Phe-NH₂D-Arg-D-Dmt-Lys-Phe-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂D-Arg-D-Dmt-D-Lys-D-Phe-NH₂ Phe-D-Arg-D-Phe-Lys-NH₂Phe-D-Arg-Phe-D-Lys-NH₂ D-Phe-D-Arg-D-Phe-D-Lys-NH₂Lys-D-Phe-Arg-Dmt-NH₂ D-Arg-Arg-Dmt-Phe-NH₂ Dmt-D-Phe-Arg-Lys-NH₂Phe-D-Dmt-Arg-Lys-NH₂ D-Arg-Dmt-Lys-NH₂ Arg-D-Dmt-Lys-NH₂D-Arg-Dmt-Phe-NH₂ Arg-D-Dmt-Arg-NH₂ Dmt-D-Arg-NH₂ D-Arg-Dmt-NH₂D-Dmt-Arg-NH₂ Arg-D-Dmt-NH₂ D-Arg-D-Dmt-NH₂ D-Arg-D-Tyr-Lys-Phe-NH₂D-Arg-Tyr-D-Lys-Phe-NH₂ D-Arg-Tyr-Lys-D-Phe-NH₂D-Arg-D-Tyr-D-Lys-D-Phe-NH₂ Lys-D-Phe-Arg-Tyr-NH₂ D-Arg-Arg-Tyr-Phe-NH₂Tyr-D-Phe-Arg-Lys-NH₂ Phe-D-Tyr-Arg-Lys-NH₂ D-Arg-Tyr-Lys-NH₂Arg-D-Tyr-Lys-NH₂ D-Arg-Tyr-Phe-NH₂ Arg-D-Tyr-Arg-NH₂ Tyr-D-Arg-NH₂D-Arg-Tyr-NH₂ D-Tyr-Arg-NH₂ Arg-D-Tyr-NH₂ D-Arg-D-Tyr-NH₂Dmt-Lys-Phe-NH₂ Lys-Dmt-D-Arg-NH₂ Phe-Lys-Dmt-NH₂ D-Arg-Phe-Lys-NH₂D-Arg-Cha-Lys-NH₂ D-Arg-Trp-Lys-NH₂ Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Lys-Phe-NH₂ D-Arg-Cha-Lys-Cha-NH₂ D-Nle-Dmt-Ahe-Phe-NH₂D-Nle-Cha-Ahe-Cha-NH₂ D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-D-Lys-Phe-NH₂Lys-Trp-D-Arg-NH₂ H-Lys-D-Phe-Arg-Dmt-NH₂ H-D-Arg-Lys-Dmt-Phe-NH₂H-D-Arg-Lys-Phe-Dmt-NH₂ H-D-Arg-Arg-Dmt-Phe-NH₂ H-D-Arg-Dmt-Phe-Lys-NH₂H-D-Arg-Phe-Dmt-Lys-NH₂ H-Dmt-D-Phe-Arg-Lys-NH₂ H-Phe-D-Dmt-Arg-Lys-NH₂H-D-Arg-Dmt-Lys-NH₂ H-D-Arg-Dmt-D-Lys-D-Phe-NH₂ H-D-Arg-Dmt-Lys-OHH-D-Arg-D-Dmt-Lys-Phe-NH₂ H-D-Arg-Dmt-OH H-D-Arg-Dmt-Phe-NH₂H-Dmt-D-Arg-NH₂ H-Phe-D-Arg-D-Phe-Lys-NH₂ H-Phe-D-Arg-Phe-D-Lys-NH₂H-D-Phe-D-Arg-D-Phe-D-Lys-NH₂ H-D-Arg-D-Dmt-D-Lys-D-Phe-NH₂H-D-Arg-Cha-Lys-NH₂ H-D-Arg-Cha-Lys-Cha-NH₂ H-Arg-D-Dmt-Lys-NH₂H-Arg-D-Dmt-Arg-NH₂ H-D-Dmt-Arg-NH₂ H-Arg-D-Dmt-NH₂ H-D-Arg-D-Dmt-NH₂6-Butyric acid CoQ0-Phe-D-Arg-Phe-Lys-NH₂ 6-Decanoic acidCoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Arg-Dmt-Phe Arg-Cha-Lys Arg-DmtArg-Dmt-Arg Arg-Dmt-Lys Arg-Dmt-Lys-Phe Arg-Dmt-Lys-Phe-Cys Arg-Dmt-PheArg-Dmt-Phe-Lys Arg-Lys-Dmt-Phe Arg-Lys-Phe-Dmt Arg-Phe-Dmt-LysArg-Phe-Lys Arg-Trp-Lys Arg-Tyr-Lys Arg-Tyr-Lys-PheD-Arg-D-Dmt-D-Lys-L-Phe-NH₂ D-Arg-D-Dmt-L-Lys-D-Phe-NH₂D-Arg-D-Dmt-L-Lys-L-Phe-NH₂ D-Arg-Dmt-D-Lys-NH₂ D-Arg-Dmt-Lys-NH₂D-Arg-Dmt-Lys-Phe-Cys D-Arg-L-Dmt-D-Lys-D-Phe-NH₂D-Arg-L-Dmt-D-Lys-L-Phe-NH₂ D-Arg-L-Dmt-L-Lys-D-Phe-NH₂ Dmt-Arg Dmt-LysDmt-Lys-Phe Dmt-Phe-Arg-Lys H-Arg-D-Dmt-Lys-Phe-NH₂H-Arg-Dmt-Lys-Phe-NH₂ H-D-Arg-2,6-dichloro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dichlorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-difluoro-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-difluorotyrosine-Lys-Phe-NH₂H-D-Arg-2,6-dimethyl-L-phenylalanine-L-Lys-L-Phe-NH₂H-D-Arg-2,6-dimethylphenylalanine-Lys-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyl-L-tyrosine-L-Lys-L-Phe-NH₂H-D-Arg-4-methoxy-2,6-dimethyltyrosine-Lys-Phe-NH₂H-D-Arg-Dmt-Lys-2,6-dimethylphenylalanine-NH₂H-D-Arg-Dmt-Lys-3-hydroxyphenylalanine-NH₂ H-D-Arg-Dmt-Lys-Phe-OHH-D-Arg-Dmt-N6-acetyllysine-Phe-NH₂ H-D-Arg-D-Phe-L-Lys-L-Phe-NH₂H-D-Arg-D-Trp-L-Lys-L-Phe-NH₂ H-D-Arg-D-Tyr-L-Lys-L-Phe-NH₂H-D-Arg-L-Dmt-L-Lys-2,6-dimethyl-L-phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-3-hydroxy-L-phenylalanine-NH₂H-D-Arg-L-Dmt-L-Lys-D-Dmt-NH₂ H-D-Arg-L-Dmt-L-Lys-D-Trp-NH₂H-D-Arg-L-Dmt-L-Lys-D-Tyr-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Dmt-NH₂H-D-Arg-L-Dmt-L-Lys-L-Trp-NH₂ H-D-Arg-L-Dmt-L-Lys-L-Tyr-NH₂H-D-Arg-L-Dmt-L-Phe-L-Lys-NH₂ H-D-Arg-L-Dmt-N6-acetyl-L-lysine-L-Phe-NH₂H-D-Arg-L-Lys-L-Dmt-L-Phe-NH₂ H-D-Arg-L-Lys-L-Phe-L-Dmt-NH₂H-D-Arg-L-Phe-L-Dmt-L-Lys-NH₂ H-D-Arg-L-Phe-L-Lys-L-Dmt-NH₂H-D-Arg-L-Phe-L-Lys-L-Phe-NH₂ H-D-Arg-L-Trp-L-Lys-L-Phe-NH₂H-D-Arg-L-Tyr-L-Lys-L-Phe-NH₂ H-D-Arg-Phe-Lys-Dmt-NH₂H-D-Arg-Tyr-Lys-Phe-NH₂ H-D-His-L-Dmt-L-Lys-L-Phe-NH₂H-D-Lys-L-Dmt-L-Lys-L-Phe-NH₂ H-Dmt-D-Arg-Lys-Phe-NH₂H-Dmt-D-Arg-Phe-Lys-NH₂ H-Dmt-Lys-D-Arg-Phe-NH₂ H-Dmt-Lys-Phe-D-Arg-NH₂H-Dmt-Phe-D-Arg-Lys-NH₂ H-Dmt-Phe-Lys-D-Arg-NH₂H-D-N2-acetylarginine-Dmt-Lys-Phe-NH₂H-D-N8-acetylarginine-Dmt-Lys-Phe-NH₂ H-L-Dmt-D-Arg-L-Lys-L-Phe-NH₂H-L-Dmt-D-Arg-L-Phe-L-Lys-NH₂ H-L-Dmt-L-Lys-D-Arg-L-Phe-NH₂H-L-Dmt-L-Lys-L-Phe-D-Arg-NH₂ H-L-Dmt-L-Phe-D-Arg-L-Lys-NH₂H-L-Dmt-L-Phe-L-Lys-D-Arg-NH₂ H-L-His-L-Dmt-L-Lys-L-Phe-NH₂H-L-Lys-D-Arg-L-Dmt-L-Phe-NH₂ H-L-Lys-D-Arg-L-Phe-L-Dmt-NH₂H-L-Lys-L-Dmt-D-Arg-L-Phe-NH₂ H-L-Lys-L-Dmt-L-Lys-L-Phe-NH₂H-L-Lys-L-Dmt-L-Phe-D-Arg-NH₂ H-L-Lys-L-Phe-D-Arg-L-Dmt-NH₂H-L-Lys-L-Phe-L-Dmt-D-Arg-NH₂ H-L-Phe-D-Arg-L-Dmt-L-Lys-NH₂H-L-Phe-D-Arg-L-Lys-L-Dmt-NH₂ H-L-Phe-L-Dmt-D-Arg-L-Lys-NH₂H-L-Phe-L-Dmt-L-Lys-D-Arg-NH₂ H-L-Phe-L-Lys-D-Arg-L-Dmt-NH₂H-L-Phe-L-Lys-L-Dmt-D-Arg-NH₂ H-Lys-D-Arg-Dmt-Phe-NH₂H-Lys-D-Arg-Phe-Dmt-NH₂ H-Lys-Dmt-D-Arg-Phe-NH₂ H-Lys-Dmt-Phe-D-Arg-NH₂H-Lys-Phe-D-Arg-Dmt-NH₂ H-Lys-Phe-Dmt-D-Arg-NH₂H-N2-acetyl-D-arginine-L-Dmt-L-Lys-L-Phe-NH₂H-N7-acetyl-D-arginine-Dmt-Lys-Phe-NH₂ H-Phe(d5)-D-Arg-Phe(d5)-Lys-NH₂H-Phe-Arg-Phe-Lys-NH₂ H-Phe-D-Arg-Dmt-Lys-NH₂ H-Phe-D-Arg-Lys-Dmt-NH₂H-Phe-D-Arg-Phe-Lys-Glu-Cys-Gly-NH₂ H-Phe-Dmt-D-Arg-Lys-NH₂H-Phe-Dmt-Lys-D-Arg-NH₂ H-Phe-Lys-D-Arg-Dmt-NH₂ H-Phe-Lys-Dmt-D-Arg-NH₂L-Arg-D-Dmt-D-Lys-D-Phe-NH₂ L-Arg-D-Dmt-D-Lys-L-Phe-NH₂L-Arg-D-Dmt-L-Lys-D-Phe-NH₂ L-Arg-D-Dmt-L-Lys-L-Phe-NH₂L-Arg-L-Dmt-D-Lys-D-Phe-NH₂ L-Arg-L-Dmt-D-Lys-L-Phe-NH₂L-Arg-L-Dmt-L-Lys-D-Phe-NH₂ L-Arg-L-Dmt-L-Lys-L-Phe-NH₂ Lys-Dmt-ArfLys-Phe Lys-Phe-Arg-Dmt Lys-Trp-Arg Phe-Arg-Dmt-Lys Phe-Arg-Phe-LysPhe-Arg-Phe-Lys-Glu-Cys-Gly Phe-Dmt-Arg-Lys Phe-Lys-Dmt Succinicmonoester CoQ0-Phe-D-Arg-Phe-Lys-NH₂ Arg-Dmt-Lys-Phe-NH₂Phe-Dmt-Arg-Lys-NH₂ Phe-Lys-Dmt-Arg-NH₂ Dmt-Arg-Lys-Phe-NH₂Lys-Dmt-Arg-Phe-NH₂ Phe-Dmt-Lys-Arg-NH₂ Arg-Lys-Dmt-Phe-NH₂Arg-Dmt-Phe-Lys-NH₂ D-Arg-Dmt-Lys-Phe-NH₂ Dmt-D-Arg-Phe-Lys-NH₂H-Phe-D-Arg Phe-Lys-Cys-NH₂ D-Arg-Dmt-Lys-Trp-NH₂ D-Arg-Trp-Lys-Trp-NH₂D-Arg-Dmt-Lys-Phe-Met-NH₂ H-D-Arg-Dmt-Lys(N^(α)Me)-Phe-NH₂H-D-Arg-Dmt-Lys-Phe(NMe)—NH₂ H-D-Arg-Dmt-Lys(N^(α)Me)-Phe(NMe)—NH₂H-D-Arg(N^(α)Me)-Dmt(NMe)-Lys(N^(α)Me)-Phe(NMe)—NH₂D-Arg-Dmt-LyS-Phe-LyS-Trp-NH₂ D-Arg-Dmt-Lys-Dmt-Lys-Trp-NH₂D-Arg-Dmt-Lys-Phe-Lys-Met-NH₂ D-Arg-Dmt-Lys-Dmt-Lys-Met-NH₂H-D-Arg-Dmt-Lys-Phe-Sar-Gly-Cys-NH₂ H-D-Arg-Ψ[CH₂—NH]Dmt-Lys-Phe-NH₂H-D-Arg-Dmt-Ψ[CH₂—NH]Lys-Phe-NH₂ H-D-Arg-Dmt-LysΨ[CH₂—NH]Phe-NH₂H-D-Arg-Dmt-Ψ[CH₂—NH]Lys-Ψ[CH₂—NH]Phe-NH₂ D-Arg-2′6′Dmt-Lys-Phe-NH2H-Phe-D-Arg-Phe-Lys-Cys-NH2Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp Dmt-D-Arg-Phe-(atn)Dap-NH₂Dmt-D-Arg-Phe-(dns)Dap-NH₂ Dmt-D-Arg-Ald-Lys-NH₂Dmt-D-Arg-Phe-Lys-Ald-NH₂ 2′,6′-dimethyltyrosine (2′6′-Dmt);dimethyltyrosine (Dmt)

In one embodiment, the aromatic-cationic peptide is defined by FormulaA:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the peptide is defined by Formula B:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the aromatic-cationic peptides of the presenttechnology have a core structural motif of alternating aromatic andcationic amino acids. For example, the peptide may be a tetrapeptidedefined by any of Formulas C to F set forth below:Aromatic-Cationic-Aromatic-Cationic  (Formula C)Cationic-Aromatic-Cationic-Aromatic  (Formula D)Aromatic-Aromatic-Cationic-Cationic  (Formula E)Cationic-Cationic-Aromatic-Aromatic  (Formula F)wherein, Aromatic is a residue selected from the group consisting of:Phe (F), Tyr (Y), and Trp (W). In some embodiments, the Aromatic residuemay be substituted with cyclohexylalanine (Cha). In some embodiments,the Cationic residue is a residue selected from the group consisting of:Arg (R), Lys (K), and His (H). In some embodiments, the Cationic residuemay be substituted with norleucine (Nle) or 2-amino-heptanoic acid(Ahe).

The amino acids of the aromatic-cationic peptides of the presenttechnology can be any amino acid. As used herein, the term “amino acid”is used to refer to any organic molecule that contains at least oneamino group and at least one carboxyl group. In some embodiments, atleast one amino group is at the α position relative to the carboxylgroup.

The amino acids may be naturally occurring. Naturally occurring aminoacids include, for example, the twenty most common levorotatory (L,)amino acids normally found in mammalian proteins, i.e., alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val).

Other naturally occurring amino acids include, for example, amino acidsthat are synthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acids ornithine and citrulline aresynthesized in mammalian metabolism during the production of urea.

The peptides useful in the present technology can contain one or morenon-naturally occurring amino acids. The non-naturally occurring aminoacids may be (L-), dextrorotatory (D-), or mixtures thereof. In someembodiments, the peptide has no amino acids that are naturallyoccurring.

Non-naturally occurring amino acids are those amino acids that typicallyare not synthesized in normal metabolic processes in living organisms,and do not naturally occur in proteins. In certain embodiments, thenon-naturally occurring amino acids useful in the present technology arealso not recognized by common proteases.

The non-naturally occurring amino acid can be present at any position inthe peptide. For example, the non-naturally occurring amino acid can beat the N terminus, the C-terminus, or at any position between theN-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenyl acetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivatives of naturally occurringamino acids may, for example, include the addition of one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of a modification of an amino acid in a peptide usefulin the present methods is the derivatization of a carboxyl group of anaspartic acid or a glutamic acid residue of the peptide. One example ofderivatization is amidation with ammonia or with a primary or secondaryamine, e.g., methylamine, ethylamine, dimethylamine or diethylamine.Another example of derivatization includes esterification with, forexample, methyl or ethyl alcohol.

Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

In some embodiments, the non-naturally occurring amino acids areresistant, and in some embodiments insensitive, to common proteases.Examples of non-naturally occurring amino acids that are resistant orinsensitive to proteases include the dextrorotatory (D-) form of any ofthe above-mentioned naturally occurring L-amino acids, as well as L-and/or D non-naturally occurring amino acids. The D-amino acids do notnormally occur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell, as used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides useful in themethods of the present technology should have less than five, less thanfour, less than three, or less than two contiguous L-amino acidsrecognized by common proteases, irrespective of whether the amino acidsare naturally or non-naturally occurring. In some embodiments, thepeptide has only D-amino acids, and no L-amino acids.

If the peptide contains protease sensitive sequences of amino acids, atleast one of the amino acids is a non-naturally-occurring D-amino acid,thereby conferring protease resistance. An example of a proteasesensitive sequence includes two or more contiguous basic amino acidsthat are readily cleaved by common proteases, such as endopeptidases andtrypsin. Examples of basic amino acids include arginine, lysine andhistidine.

It is important that the aromatic-cationic peptides have a minimumnumber of net positive charges at physiological pH in comparison to thetotal number of amino acid residues in the peptide. The minimum numberof net positive charges at physiological pH is referred to below as(p_(m)). The total number of amino acid residues in the peptide isreferred to below as (r).

The minimum number of net positive charges discussed below are all atphysiological pH. The term “physiological pH” as used herein refers tothe normal pH in the cells of the tissues and organs of the mammalianbody. For instance, the physiological pH of a human is normallyapproximately 7.4, but normal physiological pH in mammals may be any pHfrom about 7.0 to about 7.8.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2 p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, or a minimum of two net positivecharges, or a minimum of three net positive charges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally-occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

In some embodiments, carboxyl groups, especially the terminal carboxylgroup of a C-terminal amino acid, are amidated with, for example,ammonia to form the C-terminal amide. Alternatively, the terminalcarboxyl group of the C-terminal amino acid may be amidated with anyprimary or secondary amine. The primary or secondary amine may, forexample, be an alkyl, especially a branched or unbranched C₁-C₄ alkyl,or an aryl amine. Accordingly, the amino acid at the C-terminus of thepeptide may be converted to an amido, N-methylamido, N-ethylamido,N,N-dimethylamido, N,N-diethyl amido, N-methyl-N-ethylamido,N-phenylamido or N-phenyl-N-ethylamido group.

The free carboxylate groups of the asparagine, glutamine, aspartic acid,and glutamic acid residues not occurring at the C-terminus of thearomatic-cationic peptides of the present technology may also beamidated wherever they occur within the peptide. The amidation at theseinternal positions may be with ammonia or any of the primary orsecondary amines described herein.

In one embodiment, the aromatic-cationic peptide useful in the methodsof the present technology is a tripeptide having two net positivecharges and at least one aromatic amino acid. In a particularembodiment, the aromatic-cationic peptide useful in the methods of thepresent technology is a tripeptide having two net positive charges andtwo aromatic amino acids.

Aromatic-cationic peptides useful in the methods of the presenttechnology include, but are not limited to, the following peptideexamples:

TABLE 5 EXEMPLARY PEPTIDES 2′6′-Dmp-D-Arg-2′6′-Dmt-Lys-NH₂2′6′-Dmp-D-Arg-Phe-Lys-NH₂ 2′6′-Dmt-D-Arg-Phe Orn-NH₂2′6′-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoic acid)-NH₂2′6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′6′-Dmt-D-Cit-Phe Lys-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheArg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D- Arg-GlyAsp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-Arg-2′6′-Dmt-Lys-Phe-NH₂D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂ D-His-Glu-Lys-Tyr-D-Phe-ArgD-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH₂D-Tyr-Trp-Lys-NH₂Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met- NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂ Lys-D-Arg-Tyr-NH₂ Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂ Met-Tyr-D-Arg-Phe-Arg-NH₂Met-Tyr-D-Lys-Phe-Arg Phe-Arg-D-His-Asp Phe-D-Arg-2′6′-Dmt-Lys-NH₂Phe-D-Arg-His Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Phe-D-Arg-Phe-Lys-NH₂Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrThr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysTyr-D-Arg-Phe-Lys-Glu-NH₂ Tyr-D-Arg-Phe-Lys-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Tyr-His-D-Gly-MetVal-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂ D-Arg-Dmt-Lys-Trp-NH₂D-Arg-Trp-Lys-Trp-NH₂ D-Arg-Dmt-Lys-Phe-Met-NH₂H-D-Arg-Dmt-Lys(NαMe)-Phe-NH₂ H-D-Arg-Dmt-Lys-Phe(NMe)—NH₂H-D-Arg-Dmt-Lys(NαMe)-Phe(NMe)—NH₂H-D-Arg(NαMe)-Dmt(NMe)-Lys(NαMe)-Phe(NMe)—NH₂D-Arg-Dmt-Lys-Phe-Lys-Trp-NH₂ D-Arg-Dmt-Lys-Dmt-Lys-Trp-NH₂D-Arg-Dmt-Lys-Phe-Lys-Met-NH₂ D-Arg-Dmt-Lys-Dmt-Lys-Met-NH₂H-D-Arg-Dmt-Lys-Phe-Sar-Gly-Cys-NH₂ H-D-Arg-Ψ[CH2—NH]Dmt-Lys-Phe-NH₂H-D-Arg-Dmt-Ψ[CH2—NH]Lys-Phe-NH₂ H-D-Arg-Dmt-LysΨ[CH2—NH]Phe-NH₂H-D-Arg-Dmt-Ψ[CH2—NH]Lys-Ψ[CH2—NH]Phe-NH₂ D-Arg-Tyr-Lys-Phe-NH₂D-Arg-Dmt-D-Lys-Phe-NH₂ D-Arg-Dmt-Lys-D-Phe-NH₂ Phe-D-Arg-D-Phe-Lys-NH₂Phe-D-Arg-Phe-D-Lys-NH₂ D-Phe-D-Arg-D-Phe-D-Lys-NH₂Lys-D-Phe-Arg-Dmt-NH₂ D-Arg-Arg-Dmt-Phe-NH₂ Dmt-D-Phe-Arg-Lys-NH₂Phe-D-Dmt-Arg-Lys-NH₂ D-Arg-Dmt-Lys-NH₂ Arg-D-Dmt-Lys-NH₂D-Arg-Dmt-Phe-NH₂ Arg-D-Dmt-Arg-NH₂ Dmt-D-Arg-NH₂ D-Arg-Dmt-NH₂D-Dmt-Arg-NH₂ Arg-D-Dmt-NH₂ D-Arg-D-Dmt-NH₂ D-Arg-D-Tyr-Lys-Phe-NH₂D-Arg-Tyr-D-Lys-Phe-NH₂ D-Arg-Tyr-Lys-D-Phe-NH₂D-Arg-D-Tyr-D-Lys-D-Phe-NH₂ Lys-D-Phe-Arg-Tyr-NH₂ D-Arg-Arg-Tyr-Phe-NH₂Tyr-D-Phe-Arg-Lys-NH₂ Phe-D-Tyr-Arg-Lys-NH₂ D-Arg-Tyr-Lys-NH₂Arg-D-Tyr-Lys-NH₂ D-Arg-Tyr-Phe-NH₂ Arg-D-Tyr-Arg-NH₂ Tyr-D-Arg-NH₂D-Arg-Tyr-NH₂ D-Tyr-Arg-NH₂ Arg-D-Tyr-NH₂ D-Arg-D-Tyr-NH₂Dmt-Lys-Phe-NH₂ Lys-Dmt-D-Arg-NH₂ Phe-Lys-Dmt-NH₂ D-Arg-Phe-Lys-NH₂D-Arg-Cha-Lys-NH₂ D-Arg-Trp-Lys-NH₂ Dmt-Lys-D-Phe-NH₂ Dmt-Lys-NH₂Lys-Phe-NH₂ D-Arg-Cha-Lys-Cha-NH₂ D-Nle-Dmt-Ahe-Phe-NH₂D-Nle-Cha-Ahe-Cha-NH₂ Cha = cyclohexyl alanine Dmt = dimethyltyrosineDmp = dimethylphenylalanine

In some embodiments, the aromatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1.

In one embodiment, 2 p_(m) is the largest number that is less than orequal to r+1, and a may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In one embodiment, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In one embodiment, the peptides have a tyrosine residue or a tyrosinederivative at the N-terminus (i.e., the first amino acid position).Suitable derivatives of tyrosine include 2′-methyltyrosine (Mmt);2′,6′-dimethyltyrosine (2′6′-Dmt); 3′,5′-dimethyltyrosine (3′5′Dmt);N,2′,6′-trimethyltyrosine (Tmt); and 2′-hydroxy-6′-methyltyrosine (Hmt).

In one embodiment, a peptide has the formula Tyr-D-Arg-Phe-Lys-NH₂.Tyr-D-Arg-Phe-Lys-NH₂ has a net positive charge of three, contributed bythe amino acids tyrosine, arginine, and lysine and has two aromaticgroups contributed by the amino acids phenylalanine and tyrosine. Thetyrosine of Tyr-D-Arg-Phe-Lys-NH₂ can be a modified derivative oftyrosine such as in 2′,6′-dimethyltyrosine to produce the compoundhaving the formula 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂.2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ has a molecular weight of 640 and carries anet three positive charge at physiological pH.2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ readily penetrates the plasma membrane ofseveral mammalian cell types in an energy-independent manner (Zhao etal., J. Pharmacol Exp Ther., 304:425-432, 2003).

Alternatively, in some embodiments, the aromatic-cationic peptide doesnot have a tyrosine residue or a derivative of tyrosine at theN-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally-occurring or non-naturally-occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have a tyrosineresidue or a derivative of tyrosine at the N-terminus is a peptide withthe formula Phe-D-Arg-Phe-Lys-NH₂. Alternatively, the N-terminalphenylalanine can be a derivative of phenylalanine such as2′,6′-dimethylphenylalanine (2′6′-Dmp). In one embodiment, the aminoacid sequence of 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ is rearranged such that Dmtis not at the N-terminus. An example of such an aromatic-cationicpeptide is a peptide having the formula of D-Arg-2′6′-Dmt-Lys-Phe-NH₂.

Suitable substitution variants of the peptides listed herein includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group are referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group are generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that have a tyrosine residue or a tyrosinederivative at the N-terminus include, but are not limited to, thearomatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs with Mu-Opioid Activity Amino Amino Amino AminoAcid Acid Acid Acid C-Terminal Position 1 Position 2 Position 3 Position4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-Arg PheDab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-ArgPhe Lys- NH₂ NH(CH₂)₂—NH- dns 2′6′Dmt D-Arg Phe Lys- NH₂ NH(CH₂)₂—NH-atn 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-CitPhe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′DmtD-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Ahp(2- NH₂ aminoheptanoic acid)Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-ArgPhe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ TyrD-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-ArgTyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′DmtD-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt LysNH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′DmtD-Arg 2′6′Dmt Dap NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt DabNH₂ Tyr D-Lys Phe Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂Tyr D-Lys Phe Orn NH₂ 2′6′Dmt D-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe DapNH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-LysPhe Orn NH₂ 3′5′Dmt D-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′DmtD-Lys Phe Arg NH₂ Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-LysTyr Dab NH₂ Tyr D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′DmtD-Lys Tyr Orn NH₂ 2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂2′6′Dmt D-Lys 2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′DmtD-Lys 2′6′Dmt Dab NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg PhednsDap NH₂ 2′6′Dmt D-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂3′5′Dmt D-Lys 3′5′Dmt Orn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′DmtD-Lys 3′5′Dmt Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Orn Fhe Arg NH₂ TyrD-Dab Phe Arg NH₂ Tyr D-Dap Phe Arg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Orn Phe Arg NH₂ 2′6′Dmt D-Dab PheArg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂ 3′5′Dmt D-Arg Phe Arg NH₂ 3′5′DmtD-Lys Phe Arg NH₂ 3′5′Dmt D-Orn Phe Arg NH₂ Tyr D-Lys Tyr Arg NH₂ TyrD-Orn Tyr Arg NH₂ Tyr D-Dab Tyr Arg NH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′DmtD-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys 2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg NH₂ 3′5′Dmt D-Dap 3′5′Dmt ArgNH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Lys 3′5′Dmt Arg NH₂ 3′5′DmtD-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe Lys NH₂ Mmt D-Arg Phe Orn NH₂ MmtD-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂ Tmt D-Arg Phe Lys NH₂ Tmt D-ArgPhe Orn NH₂ Tmt D-Arg Phe Dab NH₂ Tmt D-Arg Phe Dap NH₂ Hmt D-Arg PheLys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-Arg Phe Dab NH₂ Hmt D-Arg Phe DapNH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys Phe Orn NH₂ Mmt D-Lys Phe Dab NH₂Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe Arg NH₂ Tmt D-Lys Phe Lys NH₂ TmtD-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂ Tmt D-Lys Phe Dap NH₂ Tmt D-LysPhe Arg NH₂ Hmt D-Lys Phe Lys NH₂ Hmt D-Lys Phe Orn NH₂ Hmt D-Lys PheDab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-Lys Phe Arg NH₂ Mmt D-Lys Phe ArgNH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab Phe Arg NH₂ Mmt D-Dap Phe Arg NH₂Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe Arg NH₂ Tmt D-Orn Phe Arg NH₂ TmtD-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂ Tmt D-Arg Phe Arg NH₂ Hmt D-LysPhe Arg NH₂ Hmt D-Orn Phe Arg NH₂ Hmt D-Dab Phe Arg NH₂ Hmt D-Dap PheArg NH₂ Hmt D-Arg Phe Arg NH₂ Dab = diaminobutyric Dap =diaminopropionic acid Dmt = dimethyltyrosine Mmt = 2′-methyltyrosine Tmt= N,2′,6′-trimethyltyrosine Hmt = 2′-hydroxy,6′-methyltyrosine dnsDap =β-dansyl-L-α,β-diaminopropionic acid atnDap =β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides that do not have a tyrosine residue or a tyrosinederivative at the N-terminus include, but are not limited to, thearomatic-cationic peptides shown in Table 7.

TABLE 7 Peptide Analogs Lacking Mu-Opioid Activity Amino Amino AminoAmino Acid Acid Acid Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 6 and 7 may be in eitherthe L- or the D-configuration.

III. Uses Of Compositions of the Present Technology

In some aspects, the methods disclosed herein provide therapies for thetreatment of medical disease or conditions and/or side effectsassociated with existing therapeutics against medical diseases orconditions comprising administering an effective amount of TBM alone orin combination with one or more aromatic-cationic peptides orpharmaceutically acceptable salts thereof, such as acetate, tartrate ortrifluoroacetate.

In another aspect, the present technology provides methods for treating,ameliorating or preventing a medical disease or condition in a subjectin need thereof, comprising administering a therapeutically effectiveamount of a composition comprising an aromatic-cationic peptide of thepresent technology conjugated to a TBM to the subject thereby treating,amelioration or preventing the medical disease or condition. Thus, forexample, one or more peptide conjugate(s) may be: (1) co-formulated andadministered or delivered alone or simultaneously in a combinedformulation with other TBMs or aromatic-cationic peptides; (2) deliveredby alternation or in parallel as separate formulations; or (3) by anyother combination therapy regimen known in the art. When delivered inalternation therapy, the methods described herein may compriseadministering or delivering the active ingredients sequentially, e.g.,in separate solution, emulsion, suspension, tablets, pills or capsules,or by different injections in separate syringes. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in simultaneoustherapy, effective dosages of two or more active ingredients areadministered together. Various sequences of intermittent combinationtherapy may also be used.

Administering combinations of aromatic peptides and TBMs can result insynergistic biological effect when administered in a therapeuticallyeffective amount to a subject suffering from a medical disease orcondition and in need of treatment. An advantage of such an approach isthat lower doses of aromatic-cationic peptide and/or TBM may be neededto prevent, ameliorate or treat a medical disease or condition in asubject. Further, potential side-effects of treatment may be avoided byuse of lower dosages of aromatic-cationic peptide and/or TBM. In someembodiments, the combination therapy comprises administering to asubject in need thereof an aromatic-cationic peptide compositioncombined with one or more TBMs. In some embodiments, the TBM and thearomatic-cationic peptide are chemically linked. In some embodiments,the TBM and the aromatic-cationic peptide are physically linked. In someembodiments, the TBM and the aromatic-cationic peptide are not linked.

Ischemia in a tissue or organ of a mammal is a multifaceted pathologicalcondition which is caused by oxygen deprivation (hypoxia) and/or glucose(e.g., substrate) deprivation. Oxygen and/or glucose deprivation incells of a tissue or organ leads to a reduction or total loss of energygenerating capacity and consequent loss of function of active iontransport across the cell membranes. Oxygen and/or glucose deprivationalso leads to pathological changes in other cell membranes, includingpermeability transition in the mitochondrial membranes. In additionother molecules, such as apoptotic proteins normally compartmentalizedwithin the mitochondria, may leak out into the cytoplasm and causeapoptotic cell death. Profound ischemia can lead to necrotic cell death.

Ischemia or hypoxia in a particular tissue or organ may be caused by aloss or severe reduction in blood supply to the tissue or organ. Theloss or severe reduction in blood supply may, for example, be due tothromboembolic stroke, coronary atherosclerosis, or peripheral vasculardisease. The tissue affected by ischemia or hypoxia is typically muscle,such as cardiac, skeletal, or smooth muscle.

The organ affected by ischemia or hypoxia may be any organ that issubject to ischemia or hypoxia. Examples of organs affected by ischemiaor hypoxia include brain, heart, kidney, and prostate. For instance,cardiac muscle ischemia or hypoxia is commonly caused by atheroscleroticor thrombotic blockages which lead to the reduction or loss of oxygendelivery to the cardiac tissues by the cardiac arterial and capillaryblood supply. Such cardiac ischemia or hypoxia may cause pain andnecrosis of the affected cardiac muscle, and ultimately may lead tocardiac failure.

Ischemia or hypoxia in skeletal muscle or smooth muscle may arise fromsimilar causes. For example, ischemia or hypoxia in intestinal smoothmuscle or skeletal muscle of the limbs may also be caused byatherosclerotic or thrombotic blockages.

Reperfusion is the restoration of blood flow to any organ or tissue inwhich the flow of blood is decreased or blocked. For example, blood flowcan be restored to any organ or tissue affected by ischemia or hypoxia.The restoration of blood flow (reperfusion) can occur by any methodknown to those in the art. For instance, reperfusion of ischemic cardiactissues may arise from angioplasty, coronary artery bypass graft, or theuse of thrombolytic drugs.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingoxLDL-induced CD36 mRNA and protein levels, and foam cell formation inmouse peritoneal macrophages. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in reducingoxLDL-induced CD36 mRNA and protein levels, and foam cell formation inmouse peritoneal macrophages.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducinginfarct volume and hemispheric swelling in a subject suffering fromacute cerebral ischemia. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing infarct volume and hemispheric swelling in a subject sufferingfrom acute cerebral ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducing thedecrease in reduced glutathione (GSH) in post-ischemic brain in asubject in need thereof. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing the decrease in reduced glutathione (GSH) in post-ischemicbrain in a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducing CD36expression in post-ischemic brain in a subject in need thereof. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing CD36 expression in post-ischemic brain in a subject in needthereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducing CD36expression in renal tubular cells after unilateral ureteral obstruction(UUO) in a subject in need thereof. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in reducing CD36 expression inrenal tubular cells after unilateral ureteral obstruction (UUO) in asubject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducing lipidperoxidation in a kidney after UUO. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in reducing lipid peroxidationin a kidney after UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingtubular cell apoptosis in an obstructed kidney after UUO. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing tubular cell apoptosis in an obstructed kidney after UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingmacrophage infiltration in an obstructed kidney induced by UUO. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing macrophage infiltration in an obstructed kidney induced by UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducinginterstitial fibrosis in an obstructed kidney after UUO. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing interstitial fibrosis in an obstructed kidney after UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingup-regulation of CD36 expression in cold storage of isolated hearts. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing up-regulation of CD36 expression in cold storage of isolatedhearts.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducing lipidperoxidation in cardiac tissue (e.g., heart) subjected to warmreperfusion after prolonged cold ischemia. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in reducing lipid peroxidationin cardiac tissue (e.g., heart) subjected to warm reperfusion afterprolonged cold ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in abolishingendothelial apoptosis in cardiac tissue (e.g., heart) subjected to warmreperfusion after prolonged cold ischemia. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in abolishing endothelialapoptosis in cardiac tissue (e.g., heart) subjected to warm reperfusionafter prolonged cold ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preservingcoronary flow in cardiac tissue (e.g., heart) subjected to warmreperfusion after prolonged cold ischemia. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in preserving coronary flow incardiac tissue (e.g., heart) subjected to warm reperfusion afterprolonged cold ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingdamage to renal proximal tubules in diabetic subjects. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inpreventing damage to renal proximal tubules in diabetic subjects.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingrenal tubular epithelial cell apoptosis in diabetic subjects. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inpreventing renal tubular epithelial cell apoptosis in diabetic subjects.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods for reducing elevated CD36expression associated with various diseases and conditions. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Examples of diseases and conditions characterized by increasedCD36 expression include, but are not limited to atherosclerosis,inflammation, abnormal angiogenesis, abnormal lipid metabolism, abnormalremoval of apoptotic cells, ischemia such as cerebral ischemia andmyocardial ischemia, ischemia-reperfusion, ureteral obstruction, stroke,Alzheimer's Disease, diabetes, diabetic nephropathy and obesity.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods for reducing CD36 expression insubjects suffering from complications of diabetes. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. Complications of diabetes include,but are not limited to, nephropathy, neuropathy, retinopathy, coronaryartery disease, and peripheral vascular disease.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods for reducing CD36 expression inremoved organs and tissues. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. The method comprises contacting the removed organ or tissue withan effective amount of a composition described herein. An organ ortissue may, for example, be removed from a donor for autologous orheterologous transplantation. Examples of organs and tissues amenable tomethods of the present technology include, but are not limited to,heart, lungs, pancreas, kidney, liver, skin, etc.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) will translocate to andaccumulate within mitochondria. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology will translocate to and accumulatewithin mitochondria.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in protectingagainst mitochondrial permeability transition (MPT) induced by Ca²⁺overload and 3-nitropropionic acid (3NP). In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in protecting againstmitochondrial permeability transition (MPT) induced by Ca²⁺ overload and3-nitropropionic acid (3NP).

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in inhibitingmitochondrial swelling and cytochrome c release. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in inhibiting mitochondrialswelling and cytochrome c release.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in protectingmyocardial contractile force during ischemia-reperfusion in cardiactissue. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inprotecting myocardial contractile force during ischemia-reperfusion incardiac tissue.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) that are administered with acardioplegic solution are useful in enhancing contractile function afterprolonged ischemia in isolated perfused cardiac tissue (e.g., heart). Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) that areadministered with a cardioplegic solution are useful in enhancingcontractile function after prolonged ischemia in isolated perfusedcardiac tissue (e.g., heart).

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in treating any disease orcondition that is associated with, for example, MPT. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Such diseases and conditions include, but are not limited to,e.g., ischemia and/or reperfusion of a tissue or organ, hypoxia,diseases and conditions of the eye, myocardial infarction and any of anumber of neurodegenerative diseases. Mammals in need of treatment orprevention of MPT are those mammals suffering from these diseases orconditions.

The methods and compositions of the present disclosure can also be usedin the treatment or prophylaxis of neurodegenerative diseases associatedwith MPT. Neurodegenerative diseases associated with MPT include, forinstance, Parkinson's disease, Alzheimer's disease, Huntington's diseaseand Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig'sdisease). The methods and compositions disclosed herein can be used todelay the onset or slow the progression of these and otherneurodegenerative diseases associated with MPT. The methods andcompositions of the present technology are useful in the treatment ofhumans suffering from the early stages of neurodegenerative diseasesassociated with MPT and in humans predisposed to these diseases.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preserving anorgan of a mammal prior to transplantation. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those including2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful in preserving an organ of amammal prior to transplantation. For example, a removed organ can besusceptible to MPT due to lack of blood flow. Therefore, thecompositions of the present disclosure can be administered to a subjectprior to organ removal, for example, and used to prevent MPT in theremoved organ.

The removed organ may be placed in a standard buffered solution, such asthose commonly used in the art. For example, a removed heart may beplaced in a cardioplegic solution containing the compositions describedherein. The concentration of compositions in the standard bufferedsolution can be easily determined by those skilled in the art. Suchconcentrations may be, for example, between about 0.1 nM to about 10 μM.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology may also be administered to a mammal taking a drug totreat a condition or disease. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

If a side effect of the drug includes MPT, mammals taking such drugswould greatly benefit from administration of the compositions disclosedherein. An example of a drug which induces cell toxicity by effectingMPT is the chemotherapy drug Adriamycin. In some embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)are useful in ameliorating, diminishing or preventing the side effectsof drugs such as adriamycin. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In certain embodiments, peptide conjugates of the presenttechnology are useful in ameliorating, diminishing or preventing theside effects of drugs such as adriamycin.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful indose-dependently scavenging H₂O₂. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in dose-dependentlyscavenging H₂O₂.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful indose-dependently inhibiting linoleic acid peroxidation induced by ABAPand reducing the rate of linoleic acid peroxidation induced by ABAP. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in dose-dependently inhibiting linoleic acidperoxidation induced by ABAP and reducing the rate of linoleic acidperoxidation induced by ABAP.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in inhibitingmitochondrial production of hydrogen peroxide, e.g., as measured byluminol chemiluminescence under basal conditions and/or upon stimulationby antimycin. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in inhibiting mitochondrial production of hydrogenperoxide, e.g., as measured by luminol chemiluminescence under basalconditions and/or upon stimulation by antimycin.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingspontaneous generation of hydrogen peroxide by mitochondria in certainstress or disease states. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful inreducing spontaneous generation of hydrogen peroxide by mitochondria incertain stress or disease states.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in inhibitingspontaneous production of hydrogen peroxide in mitochondria and hydrogenperoxide production, e.g., as stimulated by antimycin. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) are useful ininhibiting spontaneous production of hydrogen peroxide in mitochondriaand hydrogen peroxide production, e.g., as stimulated by antimycin.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasingintracellular ROS (reactive oxygen species) and increasing survival incells of a subject in need thereof. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in decreasingintracellular ROS (reactive oxygen species) and increasing survival incells of a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventing lossof cell viability in subjects suffering from a disease or conditioncharacterized by mitochondrial permeability transition. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in preventing loss of cell viability in subjectssuffering from a disease or condition characterized by mitochondrialpermeability transition.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasing thepercent of cells showing increased caspase activity in a subject in needthereof. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in decreasing the percent of cells showingincreased caspase activity in a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasing therate of ROS accumulation in a subject in need thereof. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in decreasing the rate of ROS accumulation in asubject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in inhibitinglipid peroxidation in a subject in need thereof. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in inhibiting lipidperoxidation in a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingmitochondrial depolarization and ROS accumulation in a subject in needthereof. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in preventing mitochondrial depolarization and ROSaccumulation in a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingapoptosis in a subject in need thereof. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in preventing apoptosisin a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in improvingcoronary flow in cardiac tissue (e.g., heart) subjected to warmreperfusion after prolonged (e.g., 18 hours) cold ischemia. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin improving coronary flow in cardiac tissue (e.g., heart) subjected towarm reperfusion after prolonged (e.g., 18 hours) cold ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingapoptosis in endothelial cells and myocytes in cardiac tissue (e.g.,heart) subjected to warm reperfusion after prolonged (e.g., 18 hours)cold ischemia. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin preventing apoptosis in endothelial cells and myocytes in cardiactissue (e.g., heart) subjected to warm reperfusion after prolonged(e.g., 18 hours) cold ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in improvingsurvival of pancreatic cells in a subject in need thereof. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin improving survival of pancreatic cells in a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingapoptosis and increasing viability in islet cells of pancreas insubjects in need thereof. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin reducing apoptosis and increasing viability in islet cells ofpancreas in subjects in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducingoxidative damage in pancreatic islet cells in subjects in need thereof.In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin reducing oxidative damage in pancreatic islet cells in subjects inneed thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in protectingdopaminergic cells against MPP+ toxicity in subjects in need thereof. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin protecting dopaminergic cells against MPP+ toxicity in subjects inneed thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventing lossof dopaminergic neurons in subject in need thereof. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin preventing loss of dopaminergic neurons in subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in increasingstriatal dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA(homovanillic acid) levels in subjects in need thereof. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin increasing striatal dopamine, DOPAC and HVA levels in subjects inneed thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂)are useful to reduce oxidative damage in a mammal in need thereof. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. By way of example, but not by way of limitation, mammals in needof reducing oxidative damage are those mammals suffering from a disease,condition or treatment associated with oxidative damage. Typically, theoxidative damage is caused by free radicals, such as reactive oxygenspecies (ROS) and/or reactive nitrogen species (RNS). Examples of ROSand RNS include hydroxyl radical (HO), superoxide anion radical (O₂.⁻),nitric oxide (NO.), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl),and peroxynitrite anion (ONOO⁻).

In some embodiments, a mammal in need thereof may be a mammal undergoinga treatment associated with oxidative damage. For example, the mammalmay be undergoing reperfusion. “Reperfusion” refers to the restorationof blood flow to any organ or tissue in which the flow of blood isdecreased or blocked. The restoration of blood flow during reperfusionleads to respiratory burst and formation of free radicals.

In some embodiments, a mammal in need thereof is a mammal suffering froma disease or condition associated with oxidative damage. The oxidativedamage can occur in any cell, tissue or organ of the mammal. Examples ofcells, tissues or organs affected by oxidative damage include, but arenot limited to, endothelial cells, epithelial cells, nervous systemcells, skin, heart, lung, kidney, eye and liver. For example, lipidperoxidation and an inflammatory process are associated with oxidativedamage for a disease or condition.

“Lipid peroxidation” refers to oxidative modification of lipids. Thelipids can be present in the membrane of a cell. This modification ofmembrane lipids typically results in change and/or damage to themembrane function of a cell. In addition, lipid peroxidation can alsooccur in lipids or lipoproteins exogenous to a cell. For example,low-density lipoproteins are susceptible to lipid peroxidation. Anexample of a condition associated with lipid peroxidation isatherosclerosis. Reducing oxidative damage associated withatherosclerosis is important because atherosclerosis is implicated in,for example, heart attacks and coronary artery disease.

“Inflammatory process” refers to the activation of the immune system.Typically, the immune system is activated by an antigenic substance. Theantigenic substance can be any substance recognized by the immunesystem, and include self-derived and foreign-derived substances.Non-limiting examples of diseases or conditions resulting from aninflammatory response to self-derived substances include arthritis andmultiple sclerosis. Non-limiting examples of foreign substances includeviruses and bacteria.

The virus can be any virus which activates an inflammatory process, andassociated with oxidative damage. Examples of viruses include, hepatitisA, B or C virus, human immunodeficiency virus, influenza virus, andbovine diarrhea virus. For example, hepatitis virus can elicit aninflammatory process and formation of free radicals, thereby damagingthe liver.

The bacteria can be any bacteria, and include gram-negative andgram-positive bacteria. Gram-negative bacteria containlipopolysaccharide in the bacteria wall. Examples of gram-negativebacteria include Escherichia coli, Klebsiella pneumoniae, Proteusspecies, Pseudomonas aeruginosa, Serratia, and Bacteroides. Examples ofgram-positive bacteria include pneumococci and streptococci.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or the peptide conjugates ofthe present technology (e.g., those includingD-Arg-2′6′-Dmt-Lys-Phe-NH₂) are useful in reducing oxidative damageassociated with a neurodegenerative disease or condition. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. The neurodegenerative disease can affect any cell, tissue ororgan of the central and peripheral nervous system. Non-limitingexamples of such cells, tissues and organs include, the brain, spinalcord, neurons, ganglia, Schwann cells, astrocytes, oligodendrocytes andmicroglia.

The neurodegenerative condition can be an acute condition, such as astroke or a traumatic brain or spinal cord injury. In some embodiments,the neurodegenerative disease or condition is a chronicneurodegenerative condition. In a chronic neurodegenerative condition,the free radicals can, for example, cause damage to a protein. Anexample of such a protein is amyloid precursor protein. Non-limitingexamples of chronic neurodegenerative diseases associated with damage byfree radicals include Parkinson's disease, Alzheimer's disease,Huntington's disease and Amyotrophic Lateral Sclerosis (ALS).

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in treatingpreeclampsia, diabetes, and symptoms of and conditions associated withaging, such as macular degeneration, and wrinkles. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in treatingpreeclampsia, diabetes, and symptoms of and conditions associated withaging, such as macular degeneration, and wrinkles.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in reducing oxidative damage in an organof a mammal prior to transplantation. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. For example, a removed organ, whensubjected to reperfusion after transplantation can be susceptible tooxidative damage. Therefore, the compositions of the present technologycan be used to reduce oxidative damage from reperfusion of thetransplanted organ.

The organ can be any organ suitable for transplantation. In someembodiments, the organ is a removed organ. Examples of such organsinclude, the heart, liver, kidney, lung, and pancreatic islets. In someembodiments, the removed organ is placed in a suitable medium, such asin a standard buffered solution commonly used in the art. Theconcentration of disclosed compositions in the standard bufferedsolution can be easily determined by those skilled in the art. Suchconcentrations may be, for example, between about 0.01 nM to about 10μM, about 0.1 nM to about 10 μM, about 1 μM to about 5 μM, or about 1 nMto about 100 nM.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in reducing oxidative damage in a cell inneed thereof. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Cells in need of reducing oxidative damage are generally thosecells in which the cell membrane or DNA has been damaged by freeradicals, for example, ROS and/or RNS. Examples of cells capable ofsustaining oxidative damage include, but are not limited to, pancreaticislet cells, myocytes, endothelial cells, neuronal cells, stem cells,and other cell types discussed herein.

The cells can be tissue culture cells. Alternatively, the cells may beobtained from a mammal. In one instance, the cells can be damaged byoxidative damage as a result of a cellular insult. Cellular insultsinclude, for example, a disease or condition (e.g., diabetes, etc.) orultraviolet radiation (e.g., sun, etc.). For example, pancreatic isletcells damaged by oxidative damage as a result of diabetes can beobtained from a mammal.

Due to reduction of oxidative damage, the treated cells may be capableof regenerating. Such regenerated cells may be re-introduced into themammal from which they were derived as a therapeutic treatment for adisease or condition. As mentioned above, one such condition isdiabetes.

Oxidative damage is considered to be “reduced” if the amount ofoxidative damage in a mammal, a removed organ, or a cell is decreasedafter administration of an effective amount of the compositionsdescribed herein. Typically, oxidative damage is considered to bereduced if the oxidative damage is decreased by at least about 1%, 5%,10%, at least about 25%, at least about 50%, at least about 75%, or atleast about 90%.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in regulatingoxidation state of muscle tissue. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those includingD-Arg-2′6′-Dmt-Lys-Phe-NH₂) are useful in regulating oxidation state ofmuscle tissue.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in regulatingoxidation state of muscle tissue in lean and obese human subjects. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the peptide conjugates of the presenttechnology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂) are usefulin regulating oxidation state of muscle tissue in lean and obese humansubjects.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in regulatinginsulin resistance in muscle tissue. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peptideconjugates of the present technology (e.g., those includingD-Arg-2′6′-Dmt-Lys-Phe-NH₂) are useful in regulating insulin resistancein muscle tissue.

In some embodiments, insulin resistance induced by obesity or a high-fatdiet affects mitochondrial bioenergetics. Without wishing to be bound bytheory, it is thought that the oversupply of metabolic substrates causesa reduction on the function of the mitochondrial respiratory system, andan increase in ROS production and shift in the overall redox environmentto a more oxidized state. If persistent, this leads to development ofinsulin resistance. Linking mitochondrial bioenergetics to the etiologyof insulin resistance has a number of clinical implications. Forexample, it is known that insulin resistance (NIDDM) in humans oftenresults in weight gain and, in selected individuals, increasedvariability of blood sugar with resulting metabolic and clinicalconsequences. The examples shown herein demonstrate that treatment ofmitochondrial defects with the compositions disclosed herein provides anew and surprising approach to treating or preventing insulin resistancewithout the metabolic side-effects of increased insulin.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducinginsulin resistance. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in reducing insulin resistance.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful for prophylactic and therapeutic methodsof treating a subject at risk of (or susceptible to) a disorder, or asubject having a disorder associated with insulin resistance. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Insulin resistance is generally associated with type IIdiabetes, coronary artery disease, renal dysfunction, atherosclerosis,obesity, hyperlipidemia, and essential hypertension. Insulin resistanceis also associated with fatty liver, which can progress to chronicinflammation (NASH; “nonalcoholic steatohepatitis”), fibrosis, andcirrhosis. Cumulatively, insulin resistance syndromes, including, butnot limited to diabetes, underlie many of the major causes of morbidityand death of people over age 40.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in methods for theprevention and/or treatment of insulin resistance and associatedsyndromes in a subject in need thereof. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in methods for theprevention and/or treatment of insulin resistance and associatedsyndromes in a subject in need thereof.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in improving thesensitivity of mammalian skeletal muscle tissues to insulin. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in improving the sensitivity of mammalian skeletalmuscle tissues to insulin.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingdrug-induced obesity, insulin resistance, and/or diabetes, wherein thecompound is administered with a drug that shows the side-effect ofcausing one or more of these conditions (e.g., olanzapine, Zyprexa®). Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in preventing drug-induced obesity, insulinresistance, and/or diabetes, wherein the compound is administered with adrug that shows the side-effect of causing one or more of theseconditions (e.g., olanzapine, ZYPREXA®).

Increased or decreased insulin resistance or sensitivity can be readilydetected by quantifying body weight, fasting glucose/insulin/free fattyacid, oral glucose tolerance (OGTT), in vitro muscle insulinsensitivity, markers of insulin signaling (e.g., Akt-P, IRS-P),mitochondrial function (e.g., respiration or H₂O₂ production), markersof intracellular oxidative stress (e.g., lipid peroxidation, GSH/GSSGratio or aconitase activity), or mitochondrial enzyme activity.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in methods forpreventing, in a subject, a disease or condition associated with insulinresistance in skeletal muscle tissues via modulating one or more signsor markers of insulin resistance, e.g., body weight, fastingglucose/insulin/free fatty acid, oral glucose tolerance (OGTT), in vitromuscle insulin sensitivity, markers of insulin signaling (e.g., Akt-P,IRS-P), mitochondrial function (e.g., respiration or H₂O₂ production),markers of intracellular oxidative stress (e.g., lipid peroxidation,GSH/GSSG ratio or aconitase activity), or mitochondrial enzyme activity.In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in methods for preventing, in a subject, a diseaseor condition associated with insulin resistance in skeletal muscletissues via modulating one or more signs or markers of insulinresistance, e.g., body weight, fasting glucose/insulin/free fatty acid,oral glucose tolerance (OGTT), in vitro muscle insulin sensitivity,markers of insulin signaling (e.g., Akt-P, IRS-P), mitochondrialfunction (e.g., respiration or H₂O₂ production), markers ofintracellular oxidative stress (e.g., lipid peroxidation, GSH/GSSG ratioor aconitase activity), or mitochondrial enzyme activity.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in treatingsubjects at risk for a disease that is caused or contributed to byaberrant mitochondrial function or insulin resistance. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in treating subjects at risk for a disease that iscaused or contributed to by aberrant mitochondrial function or insulinresistance.

In prophylactic applications, the compositions of the present technologyare administered to a subject susceptible to, or otherwise at risk of adisease or condition in an amount sufficient to eliminate or reduce therisk, or delay the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of prophylactic TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof),alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology can occur prior to themanifestation of symptoms characteristic of the aberrancy, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression. Depending upon the type of aberrancy, the compositions ofthe present technology will act to enhance or improve mitochondrialfunction, and can be used for treating the subject.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in methods ofmodulating insulin resistance or sensitivity in a subject fortherapeutic purposes. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in methods of modulating insulin resistance orsensitivity in a subject for therapeutic purposes.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in curing orpartially arresting the symptoms of the disease (biochemical,histological and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in curing or partially arresting the symptoms ofthe disease (biochemical, histological and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. As such, the present technology provides methods oftreating an individual afflicted with an insulin resistance-associateddisease or disorder.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in improving thehistopathological score resulting from ischemia and reperfusion. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in improving the histopathological score resultingfrom ischemia and reperfusion.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in increasing therate of ATP production after reperfusion in renal tissue followingischemia. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in increasing the rate of ATP production afterreperfusion in renal tissue following ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in improving renalmitochondrial respiration following ischemia. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in improving renalmitochondrial respiration following ischemia.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasingmedullary fibrosis in UUO. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in decreasing medullary fibrosis in UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasinginterstitial fibrosis in UUO. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in decreasinginterstitial fibrosis in UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasingtubular apoptosis in UUO. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in decreasing tubular apoptosis in UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasingmacrophage infiltration in UUO. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in decreasing macrophageinfiltration in UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in increasingtubular proliferation in UUO. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in increasing tubularproliferation in UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in decreasingoxidative damage in UUO. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in decreasing oxidative damage in UUO.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in reducing renaldysfunction caused by a radiocontrast dye. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in reducing renaldysfunction caused by a radiocontrast dye.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in protectingrenal tubules from radiocontrast dye injury. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, peptideconjugates of the present technology are useful in protecting renaltubules from radiocontrast dye injury.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) are useful in preventingrenal tubular apoptosis induced by radiocontrast dye injury. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, peptide conjugates of the presenttechnology are useful in preventing renal tubular apoptosis induced byradiocontrast dye injury.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in protecting a subject's kidney fromrenal injury. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Acute renal injury (ARI) refers to a reduction of renal functionand filtration of waste products from a patient's blood. ARI istypically characterized as including a decline of glomerular filtrationrate (GFR) to a level so low that little or no urine is formed.Therefore, substances usually eliminated by the kidney remain in thebody.

The causes of ARI may be caused by various factors, falling into threecategories: (1) pre-renal ARI, in which the kidneys fail to receiveadequate blood supply, e.g., due to reduced systemic blood pressure asin shock/cardiac arrest, or subsequent to hemorrhage; (2) intrinsic ARI,in which the failure occurs within the kidney, e.g., due to drug-inducedtoxicity; and (3) post-renal ARI, caused by impairment of urine flow outof the kidney, as in ureteral obstruction due to kidney stones orbladder/prostate cancer. ARI may be associated with any one or acombination of these categories.

An example of a condition in which kidneys fail to receive adequateblood supply to the kidney is ischemia. Ischemia is a major cause ofARI. Ischemia of one or both kidneys is a common problem experiencedduring aortic surgery, renal transplantation, or during cardiovascularanesthesia. Surgical procedures involving clamping of the aorta and/orrenal arteries, e.g., surgery for supra- and juxta-renal abdominalaortic aneurysms and renal transplantation, are also particularly liableto produce renal ischemia, leading to significant postoperativecomplications and early allograft rejection. In high-risk patientsundergoing these surgeries, the incidence of renal dysfunction has beenreported to be as high as 50%. The skilled artisan will understand thatthe above described causes of ischemia are not limited to the kidney,but may occur in other organs during surgical procedures.

Renal ischemia may be caused by loss of blood, loss of fluid from thebody as a result of severe diarrhea or burns, shock, and ischemiaassociated with storage of the donor kidney prior to transplantation. Inthese situations, the blood flow to the kidney may be reduced to adangerously low level for a time period great enough to cause ischemicinjury to the tubular epithelial cells, sloughing off of the epithelialcells into the tubular lumen, obstruction of tubular flow that leads toloss of glomerular filtration and ARI.

Subjects may also become vulnerable to ARI after receiving anesthesia,surgery, or α-adrenergic agonists because of related systemic or renalvasoconstriction. Additionally, systemic vasodilation caused byanaphylaxis, and anti-hypertensive drugs, sepsis or drug overdose mayalso cause ARI because the body's natural defense is to shut down, i.e.,vasoconstriction of non-essential organs such as the kidneys.

Accordingly, in some embodiments, a subject at risk for ARI may be asubject undergoing an interruption or reduction of blood supply or bloodpressure to the kidney. In some embodiments, these subjects may beadministered TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology prior to or simultaneously with such interruption orreduction of blood supply. Likewise, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology may be administered after the therapeutic agent to treatischemia.

Another cause of ARI includes drug-induced toxicity. For example,nephrotoxins can cause direct toxicity on tubular epithelial cells.Nephrotoxins include, but are not limited to, therapeutic drugs, e.g.,cisplatin, gentamicin, cephaloridine, cyclosporin, amphotericin,radiocontrast dye (described in further detail below), pesticides (e.g.,paraquat), and environmental contaminants (e.g., trichloroethylene anddichloroacetylene). Other examples include puromycin aminonucleoside(PAN); aminoglycosides, such as gentamicin; cephalosporins, such ascephaloridine; calcineurin inhibitors, such as tacrolimus or sirolimus.Drug-induced nephrotoxicity may also be caused by non-steroidalanti-inflammatories, antiretrovirals, anticytokines, immunosuppressants,oncological drugs, or angiotensin-converting-enzyme (ACE) inhibitors.The drug-induced nephrotoxicity may further be caused by analgesicabuse, ciprofloxacin, clopidogrel, cocaine, cox-2 inhibitors, diuretics,foscamet, gold, ifosfamide, immunoglobulin, Chinese herbs, interferon,lithium, mannitol, mesalamine, mitomycin, nitrosoureas, penicillamine,penicillins, pentamidine, quinine, rifampin, streptozocin, sulfonamides,ticlopidine, triamterene, valproic acid, doxorubicin, glycerol,cidofovir, tobramycin, neomycin sulfate, colistimethate, vancomycin,amikacin, cefotaxime, cisplatin, acyclovir, lithium, interleukin-2,cyclosporin, or indinavir.

In addition to direct toxicity on tubular epithelial cells, somenephrotoxins also reduce renal perfusion, causing injury to zones knownto have limited oxygen availability (inner medullary region). Suchnephrotoxins include amphotericin and radiocontrast dyes. Renal failurecan result even from clinically relevant doses of these drugs whencombined with ischemia, volume depletion, obstruction, or infection. Anexample is the use of radiocontrast dye in patients with impaired renalfunction. The incidence of contrast dye-induced nephropathy (CIN) is3-8% in the normal patient, but increases to 25% for patients withdiabetes mellitus. Most cases of ARI occur in patients with predisposingco-morbidities (McCombs, P. R. & Roberts, B., Surg Gynecol. Obstet.,148:175-178 (1979)).

Accordingly, in one embodiment, a subject at risk for ARI is receivingone or more therapeutic drugs that have a nephrotoxic effect. Thesubject is administered TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology prior to or simultaneously with such therapeutic agents.Likewise, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology may be administered after the therapeutic agent to treatnephrotoxicity.

In one embodiment, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject at risk for CIN, in order toprevent the condition. CIN is an important cause of acute renal failure.CIN is defined as acute renal failure occurring within 48 hours ofexposure to intravascular radiographic contrast material, and remains acommon complication of radiographic procedures.

CIN arises when a subject is exposed to radiocontrast dye, such asduring coronary, cardiac, or neuro-angiography procedures. Contrast dyeis essential for many diagnostic and interventional procedures becauseit enables doctors to visualize blocked body tissues. A creatinine testcan be used to monitor the onset of CIN, treatment of the condition, andefficacy of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology in treating or preventing CIN.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject prior to or simultaneously withthe administration of a contrast agent in order to provide protectionagainst CIN. For example, the subject may receive the compositions fromabout 1 to 2 hours, about 1 to 6 hours, about 1 to 12 hours, about 1 to24 hours, or about 1 to 48 hours prior to receiving the contrast agent.Likewise, the subject may be administered the compositions at about thesame time as the contrast agent. Moreover, administration of thecompositions to the subject may continue following administration of thecontrast agent. In some embodiments, the subject continues to receivethe compositions at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 24,and 48 hours following administration of the contrast agent, in order toprovide a protective or prophylactic effect against CIN.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject after administration of acontrast agent in order to treat CIN. For example, the subject receivesthe compositions from about 1 to 2 hours, about 1 to 6 hours, about 1 to12 hours, about 1 to 24 hours, about 1 to 48 hours, or about 1 to 72hours after receiving the contrast agent. For instance, the subject mayexhibit one or more signs or symptoms of CIN prior to receiving thecompositions of the present technology, such as increased serumcreatinine levels and/or decreased urine volume. Administration of thecompositions of the present technology improves one or more of theseindicators of kidney function in the subject compared to a controlsubject not administered the compositions.

In one embodiment of the method, a subject in need thereof may be asubject having impairment of urine flow. Obstruction of the flow ofurine can occur anywhere in the urinary tract and has many possiblecauses, including but not limited to, kidney stones or bladder/prostatecancer. UUO is a common clinical disorder associated with obstructedurine flow. It is also associated with tubular cell apoptosis,macrophage infiltration, and interstitial fibrosis. Interstitialfibrosis leads to a hypoxic environment and contributes to progressivedecline in renal function despite surgical correction. Thus, a subjecthaving or at risk for UUO may be administered TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology to prevent or treat ARI.

In yet another aspect of the present technology, a method for protectinga kidney from renal fibrosis in a mammal in need thereof is provided.The method comprises administering to the mammal an effective amount ofTBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology as described herein. The compositions described herein can beadministered to a mammal in need thereof, as described herein, by anymethod known to those skilled in the art.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods for treating ARI in a mammal inneed thereof. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. The method comprises administering to the mammal an effectiveamount of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology as described herein. The compositions described herein can beadministered to a mammal in need thereof, as described herein, by anymethod known to those skilled in the art. The methods of the presenttechnology may be particularly useful in patients with renalinsufficiency, renal failure, or end-stage renal disease attributable atleast in part to a nephrotoxicity of a drug or chemical. Otherindications may include creatinine clearance levels of lower than 97(men) and 88 (women) mL/min, or a blood urea level of 20-25 mg/dl orhigher. Furthermore, the treatment may be useful in patients withmicroalbuminuria, macroalbuminuria, and/or proteinuria levels of over 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 g or more per a 24 hour period, and/orserum creatinine levels of about 1.0, 1.5, 2.0, 2.5, 3, 3.5, 4.0, 4.5,5, 5.5, 6, 7, 8, 9, 10 mg/dl or higher.

The methods of the present technology can be used to slow or reverse theprogression of renal disease in patients whose renal function is belownormal, relative to control subjects. In some embodiments, the methodsof the present technology slow the loss of renal function. By way ofexample, but not by way of limitation, in some embodiments, loss ofrenal function is slowed by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% or more, relative to control subjects. In otherembodiments, the methods of the present technology improve the patient'sserum creatinine levels, proteinuria, and/or urinary albumin excretion.By way of example, but not by way of limitation, in some embodiments,the patient's serum creatinine levels, proteinuria, and/or urinaryalbumin excretion is improved by at least 1%, 10%, 20%, 30%, 40%, 50%,60%, 70%, or more, relative to control subjects. Non-limitingillustrative methods for assessing renal function are described hereinand, for example, in WO 01/66140.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in protecting a subject's kidney from ARIprior to transplantation. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. For example, a removed kidney can be placed in a solutioncontaining the compositions described herein. The concentration ofcompositions in the standard buffered solution can be easily determinedby those skilled in the art. Such concentrations may be, for example,between about 0.01 nM to about 10 μM, about 0.1 nM to about 10 μM, about1 μM to about 5 μM, or about 1 nM to about 100 nM.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in preventing or treating ARI and are alsoapplicable to tissue injury and organ failure in other systems besidesthe kidney. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in minimizing cell death, inflammation,and fibrosis. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods of treating a subject having atissue injury, e.g., noninfectious pathological conditions such aspancreatitis, ischemia, multiple trauma, hemorrhagic shock, andimmune-mediated organ injury. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. The tissue injury can be associatedwith, for example, aortic aneurysm repair, multiple trauma, peripheralvascular disease, renal vascular disease, myocardial infarction, stroke,sepsis, and multi-organ failure. In one aspect, the present technologyrelates to a method of treating a subject having a tissue such as fromheart, brain, vasculature, gut, liver, kidney and eye that is subject toan injury and/or ischemic event. The method includes administering tothe subject a therapeutically effective amount of TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology to provide a therapeutic orprophylactic effect.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in improving a function of one or moreorgans selected from the group consisting of: renal, lung, heart, liver,brain, pancreas, and the like. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In a particular embodiment, theimprovement in lung function is selected from the group consisting oflower levels of edema, improved histological injury score, and lowerlevels of inflammation.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in the prevention and/or treatment ofacute hepatic injury caused by ischemia, drugs (e.g., acetaminophen,alcohol), viruses, obesity (e.g., non-alcoholic steatohepatitis), andobstruction (e.g., bile duct obstruction, tumors). In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology are useful in preventing ortreating acute liver failure (ALF) in a subject. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. ALF is a clinical condition thatresults from severe and extensive damage of liver cells leading tofailure of the liver to function normally. ALF results from massivenecrosis of liver cells leading to hepatic encephalopathy and severeimpairment of hepatic function. It has various causes, such as viralhepatitis (A, B, C), drug toxicity, frequent alcohol intoxication, andautoimmune hepatitis. ALF is a very severe clinical condition with highmortality rate. Drug-related hepatotoxicity is the leading cause of ALFin the United States.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject prior to or simultaneously withthe administration of a drug or agent known or suspected to inducedhepatotoxicity, e.g., acetaminophen, in order to provide protectionagainst ALF. For example, the subject may receive the compositions fromabout 1 to 2 hours, about 1 to 6 hours, about 1 to 12 hours, about 1 to24 hours, or about 1 to 48 hours prior to receiving the drug or agent.Likewise, the subject may be administered the compositions at about thesame time as the drug or agent to provide a prophylactic effect againstALF caused by the drug or agent. Moreover, administration of thecompositions to the subject may continue following administration of thedrug or agent. In some embodiments, the subject may continue to receivethe compositions at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 24,and 48 hours following administration of the drug or agent, in order toprovide a protective or prophylactic effect.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject exhibiting one or more signs orsymptoms of ALF, including, but not limited to, elevated levels ofhepatic enzymes (transaminases, alkaline phosphatase), elevated serumbilirubin, ammonia, glucose, lactate, or creatinine. Administration ofthe compositions of the present technology improves one or more of theseindicators of liver function in the subject compared to a controlsubject not administered the compositions. The subject may receive TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology from about 1 to 2 hours, about 1 to 6 hours, about 1 to 12hours, about 1 to 24 hours, about 1 to 48 hours, or about 1 to 72 hoursafter the first signs or symptoms of ALF.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or ameliorating the local anddistant pathophysiological effects of burn injury, including, but notlimited to, hypermetabolism and organ damage. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing burn injuriesand systemic conditions associated with a burn injury. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject following a burn and after theonset of detectable symptoms of systemic injury. Thus, the term“treatment” is used herein in its broadest sense and refers to use of acomposition for a partial or complete cure of the burn and/or secondarycomplications, such as organ dysfunction and hypermetabolism.

In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject following a burn, but beforethe onset of detectable symptoms of systemic injury in order to protectagainst or provide prophylaxis for the systemic injury, such as organdamage or hypermetabolism. Thus the term “prevention” is used herein inits broadest sense and refers to a prophylactic use which completely orpartially prevents local injury to the skin or systemic injury, such asorgan dysfunction or hypermetabolism following burns. It is alsocontemplated that the compositions may be administered to a subject atrisk of receiving burns.

Burns are generally classified according to their severity and extent.First degree burns are the mildest and typically affect only theepidermis. The burn site appears red, and is painful, dry, devoid ofblisters, and may be slightly moist due to fluid leakage. Mild sunburnis typical of a first degree burn. In second degree burns, both theepidermis and dermis are affected. Blisters usually appear on the skin,with damage to nerves and sebaceous glands. Third degree burns are themost serious, with damage to all layers of the skin, includingsubcutaneous tissue. Typically there are no blisters, with the burnedsurface appearing white or black due to charring, or bright red due toblood in the bottom of the wound. In most cases, the burn penetrates thesuperficial fascia, extending into the muscle layers where arteries andveins are affected. Because of nerve damage, it is possible for the burnto be painless.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in the treatment of burns from any cause,including dry heat or cold burns, scalds, sunburn, electrical burns,chemical agents such as acids and alkalis, including hydrofluoric acid,formic acid, anhydrous ammonia, cement, and phenol, or radiation burns.In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Burns resulting from exposure to either high or low temperatureare within the scope of the present technology. The severity and extentof the burn may vary, but secondary organ damage or hypermetabolism willusually arise when the burns are very extensive or very severe (secondor third degree burns). The development of secondary organ dysfunctionor failure is dependent on the extent of the burn, the response of thepatient's immune system and other factors, such as infection and sepsis.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing organdysfunction secondary to a burn. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. The chain of physiological processeswhich lead to organ dysfunction following burns is complex. In subjectswith serious burns, release of catecholamines, vasopressin, andangiotensin causes peripheral and splanchnic bed vasoconstriction thatcan compromise the perfusion of organs remote to the injury. Myocardialcontractility also may be reduced by the release of TNF-α. Activatedneutrophils are sequestered in dermal and distant organs, such as thelung, within hours following a burn injury, resulting in the release oftoxic reactive oxygen species and proteases and producing vascularendothelial cell damage. When the integrity of pulmonary capillary andalveolar epithelia is compromised, plasma and blood leak into theinterstitial and intra-alveolar spaces, resulting in pulmonary edema. Adecrease in pulmonary function can occur in severely burned patients, asa result of bronchoconstriction caused by humoral factors, such ashistamine, serotonin, and thromboxane A2.

Subjects suffering from a burn injury are also at risk for skeletalmuscle dysfunction. While not wishing to be limited by theory,burn-induced mitochondrial skeletal muscle dysfunction is thought toresult from defects in oxidative phosphorylation (OXPHOS) viastimulation of mitochondrial production of reactive oxygen species (ROS)and the resulting damage to the mitochondrial DNA (mtDNA). In someembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology are useful in inducing ATP synthesis via a recovery of themitochondrial redox status or via the peroxisome proliferator activatedreceptor-gamma coactivator-1β, which is down-regulated as early as 6hours after a burn. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in ameliorating mitochondrial dysfunctioncaused by a burn injury. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating a wound resulting from a burninjury. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) alone or in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology may be administered systemically or topically to the wound.Burn wounds are typically uneven in depth and severity. There aretypically significant areas around the coagulated tissue where injurymay be reversible and damage mediated by the inflammatory and immunecells to the microvasculature of the skin could be prevented. In oneembodiment, the administration of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology slows or ameliorates the effects of wound contraction. Woundcontraction is the process which diminishes the size of a full-thicknessopen wound, especially a full-thickness burn. The tensions developedduring contracture and the formation of subcutaneous fibrous tissue canresult in deformity, and in particular to fixed flexure or fixedextension of a joint where the wound involves an area over the joint.Such complications are especially relevant in burn healing. No woundcontraction will occur when there is no injury to the tissue, andmaximum contraction will occur when the burn is full thickness and noviable tissue remains in the wound. In some embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology are useful in preventingprogression of a burn injury from a second degree burn to a third degreeburn. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in decreasing scarring or the formation ofscar tissue attendant the healing process at a burn site. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Scarring is the formation of fibrous tissue at sites wherenormal tissue has been destroyed. The present disclosure thus alsoincludes a method for decreasing scarring following a second or thirddegree burn. This method comprises treating an animal with a second orthird degree burn with an effective amount of TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing damage todistant organs or tissues in a subject suffering from a burn. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In particular, dysfunction or failure of the lung, liver,kidneys, and/or bowel following burns to the skin or other sites of thebody has a significant impact on morbidity and mortality. While notwishing to be limited by theory, it is believed that systemicinflammatory responses arise in subjects following burn injury, and thatit is this generalized inflammation which leads to remote tissue injurywhich is expressed as the dysfunction and failure of organs remote fromthe injury site. Systemic injury, including organ dysfunction andhypermetabolism, is typically associated with second and third degreeburns. A characteristic of the systemic injury, i.e., organ dysfunctionor hypermetabolism, is that the burn which provokes the subsequentinjury or condition does not directly affect the organ in question,i.e., the injury is secondary to the burn.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or protecting damage to livertissues secondary to a burn. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Methods for assessing liver function are well known in the artand include, but are not limited to, using blood tests for serum alanineaminotransferase (ALT) levels, alkaline phosphatase (AP), or bilirubinlevels. Methods for assessing deterioration of liver structure are alsowell known. Such methods include liver imaging (e.g., MRT, ultrasound),or histological evaluation of liver biopsy.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or protecting damage to kidneytissues secondary to a burn. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Methods for assessing kidney function are well known in the artand include, but are not limited to, using blood tests for serumcreatinine, or glomerular filtration rate. Methods for assessingdeterioration of kidney structure are also well known. Such methodsinclude kidney imaging (e.g., MRI, ultrasound), or histologicalevaluation of kidney biopsy.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in preventing or treating hypermetabolismassociated with a burn injury. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. A hypermetabolic state may beassociated with hyperglycemia, protein loss, and a significant reductionof lean body mass. Reversal of the hypermetabolic response may beaccomplished by administering TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology and by manipulating the subject's physiologic and biochemicalenvironment through the administration of specific nutrients, growthfactors, or other agents. As demonstrated in the examples, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology may be administered to a subjectsuffering from a burn in order to treat or prevent hypermetabolism.

In one aspect, the disclosure provides method for preventing in asubject, a burn injury or a condition associated with a burn injury, byadministering TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to the subject. TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology may be administered to a subject at risk of receiving burns.In prophylactic applications, pharmaceutical compositions or medicamentsof compositions of the present technology are administered to a subjectsusceptible to, or otherwise at risk of a burn injury to eliminate orreduce the risk, or delay the onset of the burn injury and itscomplications.

Another aspect of the disclosure includes methods of treating orpreventing burn injuries and associated complications in a subject fortherapeutic purposes. In therapeutic applications, compositions ormedicaments are administered to a subject already suffering from a burninjury in an amount sufficient to cure, or partially arrest, thesymptoms of the injury, including its complications and intermediatepathological phenotypes in development of the disease. TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology may be administered to a subjectfollowing a burn, but before the development of detectable symptoms of asystemic injury, such as organ dysfunction or failure, and thus the term“prevention” as used herein in its broadest sense and refers to aprophylactic use which completely or partially prevents systemic injury,such as organ dysfunction or failure or hypermetabolism following burns.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology can prevent or treat Metabolic Syndrome in mammaliansubjects. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some cases, the Metabolic Syndrome may be due to a high-fatdiet or, more generally, over-nutrition and lack of exercise. TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology may reduce one or moresigns or symptoms of Metabolic Syndrome, including, but not limited to,dyslipidemia, central obesity, blood fat disorders, and insulinresistance. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

Without wishing to be bound by theory, it is thought that loss ofmitochondrial integrity and insulin sensitivity stem from a commonmetabolic disturbance, i.e., oxidative stress. Over-nutrition,particularly from high-fat diets may increase mitochondrial reactiveoxygen species (ROS) production and overall oxidative stress, leading tothe development of metabolic syndrome. TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) or peptide conjugates ofthe present technology mitigate these effects, thereby improvingmitochondrial function in various body tissues, and improving one ormore of the risk factors associated with Metabolic Syndrome. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

The present disclosure provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) MetabolicSyndrome. Metabolic Syndrome is generally associated with type IIdiabetes, coronary artery disease, renal dysfunction, atherosclerosis,obesity, dyslipidemia, and essential hypertension. Accordingly, thepresent methods provide for the prevention and/or treatment of MetabolicSyndrome or associated conditions in a subject by administering aneffective amount of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a subject in need thereof. For example, a subject may beadministered TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to improve one or more of the factors contributing toMetabolic Syndrome. In some embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) or peptideconjugates of the present technology are useful in reducing the symptomsof Metabolic Syndrome. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In one aspect, the technology may provide a method of treating orpreventing the specific disorders associated with Metabolic Syndrome,such as obesity, diabetes, hypertension, and hyperlipidemia, in a mammalby administering TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology. In certain embodiments, the specific disorder may beobesity. In certain embodiments, the specific disorder may bedyslipidemia (i.e., hyperlipidemia).

In one embodiment, administration of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a subject exhibiting one or more conditions associatedwith Metabolic Syndrome will cause an improvement in one or more ofthose conditions (e.g., an improvement in one or more of body weight,LDL cholesterol level, HDL cholesterol level, triglyceride level, oralglucose tolerance). By way of example, but not by way of limitation, insome embodiments, a subject may exhibit at least about 5%, at leastabout 10%, at least about 20%, or at least about 50% reduction in bodyweight compared to the subject prior to receiving the TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology. By way of example, but not by wayof limitation, in some embodiments, a subject may exhibit at least about5%, at least about 10%, at least about 20%, or at least about 50%reduction in LDL cholesterol and/or at least about 5%, at least about10%, at least about 20%, or at least about 50% increase in HDLcholesterol compared to the subject prior to receiving the TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology. By way of example, but not by wayof limitation, in some embodiments, a subject may exhibit at least about5%, at least about 10%, at least about 20%, or at least about 50%reduction in some triglycerides compared to the subject prior toreceiving the TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology. By way of example, but not by way of limitation, in someembodiments, a subject may exhibit at least about 5%, at least about10%, at least about 20%, or at least about 50% improvement in oralglucose tolerance (OGTT) compared to the subject prior to receiving theTBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology. In some embodiments, the subject may show observableimprovement in more than one condition associated with MetabolicSyndrome.

In one aspect, the present technology provides a method for preventing,in a subject, a disease or condition associated with Metabolic Syndromein skeletal muscle tissues, by administering to the subject TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology that modulate one or more signs ormarkers of metabolic syndrome, e.g., body weight, serum triglycerides orcholesterol, fasting glucose/insulin/free fatty acid, oral glucosetolerance (OGTT), in vitro muscle insulin sensitivity, markers ofinsulin signaling (e.g., Akt-P, IRS-P), mitochondrial function (e.g.,respiration or H₂O₂ production), markers of intracellular oxidativestress (e.g., lipid peroxidation, GSH/GSSG ratio or aconitase activity)or mitochondrial enzyme activity. The fasting glucose/insulin/free fattyacid, oral glucose tolerance (OGTT), cholesterol and triglyceridelevels, etc. may be measured using standard clinical laboratorytechniques well-known in the art.

Subjects at risk for Metabolic Syndrome can be identified by, e.g., anyor a combination of diagnostic or prognostic assays as described herein.In prophylactic applications, pharmaceutical compositions or medicamentsof TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject susceptible to, or otherwise atrisk for a disease or condition in an amount sufficient to eliminate orreduce the risk, or delay the onset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of the prophylacticcompositions of the present technology can occur prior to themanifestation of symptoms characteristic of the aberrancy, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression. Depending upon the type of aberrancy, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) or peptideconjugates of the present technology, which act to enhance or improvemitochondrial function, can be used for treating the subject. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

Another aspect of the technology includes methods of reducing thesymptoms associated with Metabolic Syndrome in a subject for therapeuticpurposes. In therapeutic applications, compositions or medicaments ofTBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, orpartially arrest, the symptoms of the disease, including itscomplications and intermediate pathological phenotypes in development ofthe disease. As such, the present technology provides methods oftreating an individual afflicted with Metabolic Syndrome or a MetabolicSyndrome-associated disease or disorder.

The present disclosure also contemplates combination therapies of TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology with one or more agents for the treatment of blood pressure,blood triglyceride levels, or high cholesterol. Treatment for MetabolicSyndrome, obesity, insulin resistance, high blood pressure,dyslipidemia, etc., can also include a variety of other approaches,including weight loss and exercise, and dietary changes. These dietarychanges include: maintaining a diet that limits carbohydrates to 50percent or less of total calories; eating foods defined as complexcarbohydrates, such as whole grain bread (instead of white), brown rice(instead of white), sugars that are unrefined, increasing fiberconsumption by eating legumes (for example, beans), whole grains, fruitsand vegetables, reducing intake of red meats and poultry, consumption of“healthy” fats, such as those in olive oil, flaxseed oil and nuts,limiting alcohol intake, etc. In addition, treatment of blood pressure,and blood triglyceride levels can be controlled by a variety ofavailable drugs (e.g., cholesterol modulating drugs), as can clottingdisorders (e.g., via aspirin therapy) and in general, prothrombotic orproinflammatory states. If Metabolic Syndrome leads to diabetes, thereare, of course, many treatments available for this disease.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in the treatment or prevention of anophthalmic condition. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Without wishing to be limited by theory, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) or peptideconjugates of the present technology may treat or prevent ophthalmicdiseases or conditions by reducing the severity or occurrence ofoxidative damage in the eye. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In one embodiment, the ophthalmic condition is selected from thegroup consisting of: dry eye, diabetic retinopathy, cataracts, retinitispigmentosa, glaucoma, macular degeneration, choroidalneovascularization, retinal degeneration, and oxygen-inducedretinopathy.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in reducing intracellular reactive oxygenspecies (ROS) in human retinal epithelial cells (HRECs). In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in preventing the mitochondrial potentialloss of HRECs treated with high-glucose. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. The Δψm of HRECs can be measured byflow cytometry after JC-1 fluorescent probe staining. High glucose (30mM) treatment results in a rapid loss of mitochondrial membranepotential of the cultured HRECs. In some embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology are useful in increasingΔψm in high glucose treated HRECs. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in reducing the elevated expression ofcaspase-3 in high glucose-treated HRECs. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology are useful in increasingthe expression of Trx2 in the high glucose-treated HRECs. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) alone or in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology will have no adverse effects on the viability of primaryhuman retinal pigment epithelial (RPE) cells.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) anophthalmic disease or condition. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. Accordingly, the present methodsprovide for the prevention and/or treatment of an ophthalmic conditionin a subject by administering an effective amount of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology to a subject in need thereof. Forexample, a subject can be administered compositions comprising TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology to improve one or more of thefactors contributing to an ophthalmic disease or condition.

One aspect of the present technology includes methods of reducing anophthalmic condition in a subject for therapeutic purposes. Intherapeutic applications, compositions or medicaments comprising TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject known to have or suspected ofhaving a disease, in an amount sufficient to cure, or at partiallyarrest/reduce, the symptoms of the disease, including complications andintermediate pathological phenotypes in development of the disease. Assuch, the disclosure provides methods of treating an individualafflicted with an ophthalmic condition. In some embodiments, thetechnology provides a method of treating or preventing specificophthalmic disorders, such as diabetic retinopathy, cataracts, retinitispigmentosa, glaucoma, choroidal neovascularization, retinaldegeneration, and oxygen-induced retinopathy, in a mammal byadministering TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing diabeticretinopathy in a subject. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Diabetic retinopathy is characterized by capillarymicroaneurysms and dot hemorrhaging. Thereafter, microvascularobstructions cause cotton wool patches to form on the retina. Moreover,retinal edema and/or hard exudates may form in individuals with diabeticretinopathy due to increased vascular hyperpermeability. Subsequently,neovascularization appears and retinal detachment is caused by tractionof the connective tissue grown in the vitreous body. Iris rubeosis andneovascular glaucoma may also occur which, in turn, can lead toblindness. The symptoms of diabetic retinopathy include, but are notlimited to, difficulty reading, blurred vision, sudden loss of vision inone eye, seeing rings around lights, seeing dark spots, and/or seeingflashing lights.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing cataracts in asubject. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Cataracts are a congenital or acquired disease characterized bya reduction in natural lens clarity. Individuals with cataracts mayexhibit one or more symptoms, including, but not limited to, cloudinesson the surface of the lens, cloudiness on the inside of the lens, and/orswelling of the lens. Typical examples of congenital cataract-associateddiseases are pseudo-cataracts, membrane cataracts, coronary cataracts,lamellar cataracts, punctuate cataracts, and filamentary cataracts.Typical examples of acquired cataract-associated diseases are geriatriccataracts, secondary cataracts, browning cataracts, complicatedcataracts, diabetic cataracts, and traumatic cataracts. Acquiredcataracts are also inducible by electric shock, radiation, ultrasound,drugs, systemic diseases, and nutritional disorders. Acquired cataractsfurther include postoperative cataracts.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing retinitispigmentosa in a subject. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Retinitis pigmentosa is a disorder that is characterized by rodand/or cone cell damage. The presence of dark lines in the retina istypical in individuals suffering from retinitis pigmentosa. Individualswith retinitis pigmentosa also present with a variety of symptomsincluding, but not limited to, headaches, numbness or tingling in theextremities, light flashes, and/or visual changes. See, e.g.,Heckenlively, et al., Am. J. Ophthalmol. 105(5):504-511 (1988).

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing glaucoma in asubject. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Glaucoma is a genetic disease characterized by an increase inintraocular pressure, which leads to a decrease in vision. Glaucoma mayemanate from various ophthalmologic conditions that are already presentin an individual, such as, wounds, surgery, and other structuralmalformations. Although glaucoma can occur at any age, it frequentlydevelops in elderly individuals and leads to blindness. Glaucomapatients typically have an intraocular pressure in excess of 21 mm Hg.However, normal tension glaucoma, where glaucomatous alterations arefound in the visual field and optic papilla, can occur in the absence ofsuch increased intraocular pressures, i.e., greater than 21 mm Hg.Symptoms of glaucoma include, but are not limited to, blurred vision,severe eye pain, headache, seeing haloes around lights, nausea, and/orvomiting.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing maculardegeneration in a subject. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Macular degeneration is typically an age-related disease. Thegeneral categories of macular degeneration include wet, dry, andnon-aged related macular degeneration. Dry macular degeneration, whichaccounts for about 80-90 percent of all cases, is also known asatrophic, nonexudative, or drusenoid macular degeneration. With drymacular degeneration, drusen typically accumulate beneath the retinalpigment epithelium tissue. Vision loss subsequently occurs when druseninterfere with the function of photoreceptors in the macula. Symptoms ofdry macular generation include, but are not limited to, distortedvision, center-vision distortion, light or dark distortion, and/orchanges in color perception. Dry macular degeneration can result in thegradual loss of vision.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing choroidalneovascularization in a subject. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. Choroidal neovascularization (CNV) isa disease characterized by the development of new blood vessels in thechoroid layer of the eye. The newly formed blood vessels grow in thechoroid, through the Bruch membrane, and invade the sub-retinal space.CNV can lead to the impairment of sight or complete loss of vision.Symptoms of CNV include, but are not limited to, seeing flickering,blinking lights, or gray spots in the affected eye or eyes, blurredvision, distorted vision, and/or loss of vision.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing retinaldegeneration in a subject. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Retinal degeneration is a genetic disease that relates to thebreak-down of the retina. Retinal tissue may degenerate for variousreasons, such as, artery or vein occlusion, diabetic retinopathy,retinopathy of prematurity, and/or retrolental fibroplasia. Retinaldegradation generally includes retinoschisis, lattice degeneration, andis related to progressive macular degeneration. The symptoms of retinadegradation include, but are not limited to, impaired vision, loss ofvision, night blindness, tunnel vision, loss of peripheral vision,retinal detachment, and/or light sensitivity.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in treating or preventing oxygen-inducedretinopathy in a subject. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Oxygen-induced retinopathy (OIR) is a disease characterized bymicrovascular degeneration. OIR is an established model for studyingretinopathy of prematurity. OIR is associated with vascular cell damagethat culminates in abnormal neovascularization. Microvasculardegeneration leads to ischemia which contributes to the physical changesassociated with OIR. Oxidative stress also plays an important role inthe development of OIR where endothelial cells are prone to peroxidativedamage. Pericytes, smooth muscle cells, and perivascular astrocytes,however, are generally resistant to peroxidative injury. See, e.g.,Beauchamp, et al., J. Appl. Physiol. 90:2279-2288 (2001). OIR, includingretinopathy of prematurity, is generally asymptomatic. However, abnormaleye movements, crossed eyes, severe nearsightedness, and/or leukocoria,can be a sign of OIR or retinopathy of prematurity.

In one aspect, the present technology provides a method for preventingan ophthalmic condition in a subject by administering to the subject aneffective amount of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology that modulates one or more signs or markers of an ophthalmiccondition. Subjects at risk for an ophthalmic condition can beidentified by, e.g., any or a combination of diagnostic or prognosticassays as described herein. In prophylactic applications, pharmaceuticalcompositions or medicaments comprising TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) alone or in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject susceptible to, or otherwise atrisk of a disease or condition in an amount sufficient to eliminate orreduce the risk, or delay the onset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of the prophylacticcompositions of the present technology can occur prior to themanifestation of symptoms characteristic of the aberrancy, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression. Depending upon the type of aberrancy, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) or peptideconjugates of the present technology act to enhance or improvemitochondrial function or reduce oxidative damage, and can be used fortreating the subject. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful for both prophylactic and therapeuticmethods of treating a subject having or at risk of (susceptible to)heart failure. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Accordingly, the present methods provide for the preventionand/or treatment of heart failure in a subject by administering aneffective amount of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a subject in need thereof. In particular embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) or peptide conjugates of the present technology are used totreat or prevent heart failure by enhancing mitochondrial function incardiac tissues. In other embodiments, TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

One aspect of the technology includes methods of treating heart failurein a subject for therapeutic purposes. In therapeutic applications,compositions or medicaments comprising TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) alone or in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, orpartially arrest, the symptoms of the disease, including itscomplications and intermediate pathological phenotypes in development ofthe disease. As such, the present technology provides methods oftreating an individual afflicted with heart failure.

Subjects suffering from heart failure can be identified by any or acombination of diagnostic or prognostic assays known in the art. Forexample, typical symptoms of heart failure include shortness of breath(dyspnea), fatigue, weakness, difficulty breathing when lying flat, andswelling of the legs, ankles, or abdomen (edema). The subject may alsobe suffering from other disorders including coronary artery disease,systemic hypertension, cardiomyopathy or myocarditis, congenital heartdisease, abnormal heart valves or valvular heart disease, severe lungdisease, diabetes, severe anemia hyperthyroidism, arrhythmia ordysrhythmia and myocardial infarction. The primary signs of congestiveheart failure are: cardiomegaly (enlarged heart), tachypnea (rapidbreathing; occurs in the case of left side failure) and hepatomegaly(enlarged liver; occurs in the case of right side failure). Acutemyocardial infarction (“AMI”) due to obstruction of a coronary artery isa common initiating event that can lead ultimately to heart failure.However, a subject that has AMI does not necessarily develop heartfailure. Likewise, subjects that suffer from heart failure do notnecessarily suffer from an AMI.

In one aspect, the present technology provides a method of treatinghypertensive cardiomyopathy by administering an effective amount of TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a subject in need thereof. As hypertensive cardiomyopathyworsens, it can lead to congestive heart failure. Subjects sufferingfrom hypertensive cardiomyopathy can be identified by any or acombination of diagnostic or prognostic assays known in the art. Forexample, typical symptoms of hypertensive cardiomyopathy includehypertension (high blood pressure), cough, weakness, and fatigue.Additional symptoms of hypertensive cardiomyopathy include leg swelling,weight gain, difficulty breathing when lying flat, increasing shortnessof breath with activity, and waking in the middle of the night short ofbreath.

In one aspect, the present technology provides a method for preventingheart failure in a subject by administering to the subject TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology that prevent the initiation orprogression of the infarction. Subjects at risk for heart failure can beidentified by, e.g., any or a combination of diagnostic or prognosticassays as described herein. In prophylactic applications, pharmaceuticalcompositions or medicaments of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject susceptible to, or otherwise atrisk of a disease or condition in an amount sufficient to eliminate orreduce the risk, or delay the onset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of prophylactic TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology can occur prior to themanifestation of symptoms characteristic of the aberrancy, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in reducing activation of p38 MAPK andapoptosis in response to Ang II. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in ameliorating myocardial performanceindex (MPI) in Gαq mice. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in preventing an increase in normalizedheart weight. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in promoting normalized lung weight in Gαqmice. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods for treating, ameliorating orreversing left ventricular stiffening, ventricular wall thickening,abnormal left ventricular relaxation and filling, LV remodeling, cardiacmyocyte hypertrophy, inflammation, other abnormal left ventricularfunction, myocardial fibrosis, and/or myocardial extracellular matrixaccumulation, and preventing progression to diastolic heart failure. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Moreover, it is proposed that these improvements in diastolicheart disease (DHD) pathology will have a resultant positive effect onthe health of the individuals by reducing complications of myocardialfibrosis and left ventricular stiffness, including the development ofdiastolic dysfunction and diastolic heart failure.

In some embodiments, an effective dose of TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology, can be administered via a varietyof routes including, but not limited to, e.g., parenteral via anintravenous infusion given as repeated bolus infusions or constantinfusion, intradermal injection, subcutaneously given as repeated bolusinjection or constant infusion, or oral administration.

In certain embodiments, an effective parenteral dose (givenintravenously, intraperitoneally, or subcutaneously) of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology to an experimental animal is withinthe range of 2 mg/kg up to 160 mg/kg body weight, or 10 mg/kg, or 30mg/kg, or 60 mg/kg, or 90 mg/kg, or 120 mg/kg body weight.

In some embodiments, an effective parenteral dose (given intravenously,intraperitoneally, or subcutaneously) of TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology to an experimental animal can beadministered three times weekly, twice weekly, once weekly, once everytwo weeks, once monthly, or as a constant infusion.

In certain embodiments, an effective parental dose (given intravenouslyor subcutaneously) of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a human subject is within the range of 0.5 mg/kg up to 25mg/kg body weight, or 1 mg/kg, or 2 mg/kg, or 5 mg/kg or 7.5 mg/kg, or10 mg/kg body weight, or 15 mg/kg body weight.

In some embodiments, an effective parental dose (given intravenously orsubcutaneously) of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a human subject can be administered three times weekly,twice weekly, once weekly, once every two weeks, once monthly, or as aconstant infusion.

In some embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change inserum biomarkers, e.g., of at least 1-10% in the level of the serumbiomarkers of DHD including, but not limited to, e.g., hyaluronic acid,type I collagen carboxyterminal telopeptide (ICTP), and other breakdownproducts of collagens, titin, troponin I, troponin T and othercytoskeletal cellular proteins, matrix metalloprotease-9, tissueinhibitor of matrix metalloproteases 2 (TIMP2) and other myocardialderived collagen and matrix proteases. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. These compounds and biomarkers may bemeasured in serum or myocardial tissue using immunoassays and the levelscorrelated with severity of disease and treatment.

In some embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change of atleast 1-10% in serum biomarkers of DHD including, but not limited to,e.g., reactive oxygen products of lipid or protein origin, coenzyme Qreduced or oxidized forms, and lipid molecules or conjugates. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. These biomarkers can be measured by various means includingimmunoassays and electrophoresis and their levels correlated withseverity of disease and treatment.

In some embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change of atleast 1-10% in serum biomarkers of DHD including, but not limited to,e.g., cytokines that include but are not limited to TNF-α, TGF-β, IL-6,IL-8, or monocyte chemoattractant protein 1 (MCP-1) osteopontin, or ametabolic profile of serum components that is indicative of DHDoccurrence or severity (these include serum and urine markers). In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. A profile of one or more of these cytokines, as measured byimmunoassay or proteomic assessment by LC mass spec, may provide anassessment of activity of the disease and a marker to follow in therapyof the disease.

In some embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change of atleast 1-10% in the clinical manifestations of DHD including, but notlimited to, e.g., clinical testing of stage and severity of the disease,clinical signs and symptoms of disease, and medical complications. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Clinical testing of stage and severity of DHD include, but arenot limited to, e.g., hematologic testing (including, but not limitedto, e.g., red blood cell count and morphology, white blood cell countand differential and morphology, platelet count and morphology), serumor plasma lipids including, but not limited to, e.g., triglycerides,cholesterol, fatty acids, lipoprotein species and lipid peroxidationspecies, serum or plasma enzymes (including, but not limited to, e.g.,aspartate transaminase (AST), creatine kinase (CK-MB), lactatedehydrogenase (LDH) and isoforms, serum or plasma brain natriureticpeptide (BNP), cardiac troponins, and other proteins indicative of heartfailure or damage, including ischemia or tissue necrosis, serum orplasma electrolytes (including, but not limited to, e.g., sodium,potassium, chloride, calcium, phosphorous), coagulation profileincluding, but not limited to, e.g., prothrombin time (PT), partialthromoplastin time (PTT), specific coagulation factor levels, bleedingtime and platelet function. Clinical testing also includes but is notlimited to non-invasive and invasive testing that assesses thearchitecture, structural integrity or function of the heart including,but not limited to, e.g., computerized tomography (CT scan), ultrasound(US), ultrasonic elastography (including, but not limited to, e.g.,(Time Harmonic Elastography) or other measurements of the elasticity ofheart tissue, magnetic resonance scanning or spectroscopy, percutaneousor skinny needle or transjugular liver biopsy and histologicalassessment (including, but not limited to, e.g., staining for differentcomponents using affinity dyes or immunohistochemistry), or othernon-invasive or invasive tests that may be developed for assessingseverity of DHD in the heart tissue.

In some embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change of atleast 1-10% in the pathophysiologic spectrum of DHD which includeshistopathological findings on heart biopsy that include but are notlimited to evidence of myocyte hypertrophy, perivascular andinterstitial fibrosis, extracellular matrix accumulation, collagendeposition, inflammatory cell infiltrates (including, but not limitedto, e.g., lymphocytes and various subsets of lymphocytes andneutrophils), changes in endothelial cells, and methods that combinevarious sets of observations for grading the severity of DHD. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In certain embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change of atleast 1-10% in the pathophysiologic spectrum of DHD which includescardiac imaging measurements and analysis, that include but are notlimited to Doppler and Tissue Doppler echocardiographic measures of leftventricular isovolumetric relaxation time (IVRT), E/A ratio (transmitralblood flow), pulmonary vein flow, E wave deceleration time, pulmonaryvein A-wave reversal velocity, pulmonary artery systolic pressure, leftventricular mass, left atrial volume, and E/E′ ratio (ration transmitralblood flow in early diastole with mitral annular velocity during earlydiastole, which characterizes left ventricular diastolic pressures). Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Speckle Tracking and ultrasound imaging methods may also beused.

In some embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, results in a change of atleast 1-10% in clinical signs and symptoms of disease include dyspnea,pulmonary congestion, pulmonary edema, flash pulmonary edema, pulmonaryhypertension, tachypnea, orthopnea, lung crepitations, coughing,fatigue, sleep disturbance, peripheral edema, and other organ edema. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. The symptoms of diastolic heart failure progress quickly andbecome sufficiently severe to warrant placement on a hearttransplantation list or receiving a heart transplantation.

In certain embodiments, a therapeutically effective dose of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology, has an effect on DHDand/or fibrosis in the absence of any effect on whole blood glucose inpatients with diabetes or serum lipids in patients with elevated serumlipids. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, a therapeutically effective dose of TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) or peptide conjugates of the present technology, results in areduction of at least 1-10% in the level of galectin-3 in heart tissueor serum. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods of treating a subject havingdiastolic heart disease, diastolic dysfunction, diastolic heart failure,left ventricular stiffening, ventricular wall thickening, abnormal leftventricular relaxation and filling, LV remodeling, cardiac myocytehypertrophy, myocardial fibrosis, inflammation, and/or myocardialextracellular matrix accumulation. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In some embodiments, the method comprises the steps of obtaining acomposition for parenteral or enteral administration comprising TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates in an acceptable pharmaceutical carrier; administering to asubject an effective dose of the composition for parenteraladministration, the subject having diastolic heart disease, diastolicdysfunction, diastolic heart failure, left ventricular stiffening,ventricular wall thickening, abnormal left ventricular relaxation andfilling, LV remodeling, cardiac myocyte hypertrophy, myocardialfibrosis, inflammation, and/or myocardial extracellular matrixaccumulation.

In some embodiments, administration of a therapeutically effective doseof TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) or peptide conjugates of the present technology to a subject inneed thereof, results in the prevention, amelioration, or treatment ofdiastolic heart disease, diastolic dysfunction, diastolic heart failure,left ventricular stiffening, ventricular wall thickening, abnormal leftventricular relaxation and filling, LV remodeling, cardiac myocytehypertrophy, myocardial fibrosis, inflammation, and/or myocardialextracellular matrix accumulation. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In certain embodiments, administration of a therapeutically effectivedose of TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) or peptide conjugates of the present technology to asubject in need thereof, can result in reduction of at least one gradein severity of diastolic heart disease scoring systems, reduction of thelevel of serum markers of diastolic heart disease, reduction ofdiastolic heart disease activity or reduction in the medicalconsequences of diastolic heart disease. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In certain embodiments, administration of a therapeutically effectivedose of TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) or peptide conjugates of the present technology to asubject in need thereof, can result in the reduction of cardiac tissuecell ballooning as determined from cardiac tissue histological sectionby assessment of swelling of cardiac tissue cells indicating toxicityand inability to regulate cellular volume. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the cardiactissue cell ballooning is reduced by at least 1-10% compared to theextent of swelling present prior to administration of the composition.

In some embodiments, administration of a therapeutically effective doseof TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) or peptide conjugates of the present technology to a subject inneed thereof, can result in the reduction in the infiltration ofinflammatory cells in cardiac tissue histological specimens, as assessedby the number of neutrophils and lymphocytes. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the infiltrationof inflammatory cells in cardiac tissue histological specimens isreduced by at least 1-10%, compared to the percentage of inflammatorycells observed prior to administration of the composition.

In certain embodiments, administration of a therapeutically effectivedose of TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) or peptide conjugates of the present technology to asubject in need thereof, can result in the reduction of accumulation ofcollagen in the heart as determined by quantitative analysis of SiriusRed staining of cardiac tissue histological sections. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the reduction of accumulation of collagenin the heart is reduced by at least 1-5% compared to the percentage ofcardiac tissue staining positive for Sirius red (indicating collagen)prior to administration of the composition.

In certain embodiments, administration of a therapeutically effectivedose of TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) or peptide conjugates of the present technology to asubject in need thereof, can result in the reduction in the level of theserum markers of diastolic heart disease activity. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the serummarkers of diastolic heart disease activity can include, but are notlimited to, serum levels of brain natriuretic peptide (BNP), cardiactroponin T, degraded titan, type I collagen telopeptide, serum levels ofcoenzyme Q reduced or oxidized, or a combination of other serum markersof diastolic heart disease activity known in the art.

In certain embodiments, administration of a therapeutically effectivedose of TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) or peptide conjugates of the present technology to asubject in need thereof, can result in the reduction of cardiac tissuefibrosis, thickening, stiffness, or extracellular matrix accumulationbased on evidence comprising a reduction of the level of the biochemicalmarkers of fibrosis, non-invasive testing of cardiac tissue fibrosis,thickening, stiffness, or extracellular matrix accumulation or cardiactissue histologic grading of fibrosis, thickening, stiffness, orextracellular matrix accumulation. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In some embodiments, administration of a therapeutically effective doseof TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) or peptide conjugates of the present technology to a subject inneed thereof, can result in the reduction of at least one grade inseverity of diastolic heart disease grading scoring systems including,but not limited to, e.g., the Mayo Clinic Doppler echocardiographicdiastolic dysfunction I-IV classification system (Nishimura R A, et al.,J Am Coll Cardiol. 30:8-18 (1997)), or the Canadian consensusrecommendations for echocardiographic measurement of diastolicdysfunction (Rakowski H., et al., J Am Soc Echocardiogr 9:736-60(1996)). In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In certain embodiments, administration of a therapeutically effectivedose of TBMs (or derivatives, analogues, or pharmaceutically acceptablesalts thereof) or peptide conjugates of the present technology to asubject in need thereof, can result in the reduction in the medicalconsequences of diastolic heart disease such as pulmonary congestion,pulmonary edema, flash pulmonary edema, pulmonary hypertension,tachypnea, dyspnea, orthopnea, lung crepitations, and other edema. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, the efficacy of a composition comprising TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology for parenteral administration canbe determined by administering the composition to animal models ofdiastolic heart disease, including, but not limited to, e.g., micesubjected to aortic constriction or Dahl salt-sensitive hypertensiverats. In some embodiments, administration of the TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) or peptideconjugate composition to animal models of diastolic heart disease canresult in at least a 1-5% reduction in heart infiltration byinflammatory cells or at least a 1-5% reduction in heart collagencontent as determined by morphometric quantification. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some aspects, the present technology relates to compositions havingTBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology for the treatment of diastolic heart disease, diastolicdysfunction, diastolic heart failure, left ventricular stiffening,ventricular wall thickening, abnormal left ventricular relaxation andfilling, LV remodeling, cardiac myocyte hypertrophy, myocardialfibrosis, inflammation, and/or myocardial extracellular matrixaccumulation.

Other aspects of the present technology relate to the use of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology, in the manufacture of apharmaceutical composition for the treatment of diastolic heart disease,diastolic dysfunction, diastolic heart failure, left ventricularstiffening, ventricular wall thickening, abnormal left ventricularrelaxation and filling, LV remodeling, cardiac myocyte hypertrophy,myocardial fibrosis, inflammation, and/or myocardial extracellularmatrix accumulation.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) vesselocclusion injury, ischemia-reperfusion injury, or cardiacischemia-reperfusion injury. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Accordingly, the present methods provide for the preventionand/or treatment of vessel occlusion injury, ischemia-reperfusioninjury, or cardiac ischemia-reperfusion injury in a subject byadministering an effective amount of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a subject in need thereof or of a subject having acoronary artery bypass graft (CABG) procedure.

In one aspect, the present technology provides a method for preventingvessel occlusion injury in a subject by administering to the subjectTBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology that prevent the initiation or progression of the condition.Subjects at risk for vessel occlusion injury can be identified by, e.g.,any or a combination of diagnostic or prognostic assays as describedherein. In prophylactic applications, pharmaceutical compositions ormedicaments comprising TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject susceptible to, or otherwise atrisk of a disease or condition in an amount sufficient to eliminate orreduce the risk, or delay the onset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of prophylactic TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology can occur prior to themanifestation of symptoms characteristic of the aberrancy, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression. In some embodiments, the compositions are administered insufficient amounts to prevent renal or cerebral complications from CABG.

Another aspect of the present technology includes methods of treatingvessel occlusion injury or ischemia-reperfusion injury in a subject. Intherapeutic applications, compositions or medicaments comprising TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates are administered toa subject suspected of, or already suffering from such a disease in anamount sufficient to cure, or partially arrest, the symptoms of thedisease, including its complications and intermediate pathologicalphenotypes in development of the disease. As such, the technologyprovides methods of treating an individual afflicted withischemia-reperfusion injury or treating an individual afflicted withcardiac ischemia-reperfusion injury by administering an effective amountof TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology and performing a CABG procedure.

The present technology also potentially relates to compositions andmethods for the treatment or prevention of ischemia-reperfusion injuryassociated with AMI and organ transplantation in mammals. In general,the methods and compositions include one or more TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology or pharmaceutically acceptablesalts thereof.

In some aspects, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology are used in methods for treating AMI injury in mammals. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some aspects, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology are used in methods for ischemia and/or reperfusion injurymammals. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some aspects, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology are used in methods for the treatment, prevention oralleviation of symptoms of cyclosporine-induced nephrotoxicity injury inmammals. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some aspects, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology are used in methods for performing revascularizationprocedures in mammals. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In one embodiment, the revascularization procedure is selected from thegroup consisting of: percutaneous coronary intervention; balloonangioplasty; insertion of a bypass graft; insertion of a stent; anddirectional coronary atherectomy. In some embodiments, therevascularization procedure comprises removal of the occlusion. In someembodiments, the revascularization procedure comprises administration ofone or more thrombolytic agents. In some embodiments, the one or morethrombolytic agents are selected from the group consisting of: tissueplasminogen activator; urokinase; prourokinase; streptokinase; anacylated form of plasminogen; acylated form of plasmin; and acylatedstreptokinase-plasminogen complex.

In another aspect, the present disclosure provides a method of coronaryrevascularization comprising: (a) administering simultaneously,separately or sequentially an effective amount of (i) TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology or a pharmaceutically acceptablesalt and (ii) an additional active agent; and (b) performing a coronaryartery bypass graft procedure on the subject. In some embodiments, theadditional active agent comprises cyclosporine or a cyclosporinederivative or analogue.

In another aspect, the present disclosure provides a method of coronaryrevascularization comprising: (a) administering to a mammalian subject atherapeutically effective amount of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology or a pharmaceutically acceptable salt thereof; (b)administering to the subject a therapeutically effective amount ofcyclosporine or a cyclosporine derivative or analogue; and (c)performing a coronary artery bypass graft procedure on the subject.

In one aspect, the present technology provides a method for preventingAMI injury in a subject by administering to the subject TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology and cyclosporine that prevent theinitiation or progression of the condition. In prophylacticapplications, pharmaceutical compositions or medicaments comprising TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology and cyclosporine are administered to a subject susceptibleto, or otherwise at risk of a disease or condition in an amountsufficient to eliminate or reduce the risk, or delay the onset of thedisease, including biochemical, histologic and/or behavioral symptoms ofthe disease, its complications and intermediate pathological phenotypespresenting during development of the disease. Administration ofprophylactic TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology and cyclosporine can occur prior to the manifestation ofsymptoms characteristic of the aberrancy, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology, and cyclosporine are useful in protecting kidneysfrom ARI. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Another aspect of the technology includes methods of treatingischemia in any organ or tissue. Accordingly, in some embodiments, suchischemia can be treated, prevented, ameliorated (e.g., the severity ofischemia is decreased) by the administration of TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology or a pharmaceutically acceptablesalt thereof, such as acetate, tartrate, or trifluoroacetate salt, andan active agent, such as cyclosporine or a derivative or analoguethereof.

Another aspect of the present technology includes methods for preventingor ameliorating cyclosporine-induced nephrotoxicity. For example, insome embodiments, a pharmaceutical composition or medicament comprisingTBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology is administered to a subject presenting with or at risk ofcyclosporine-induced nephrotoxicity. For example, in some embodiments, asubject receiving cyclosporine, e.g., as an immunosuppressant after anorgan or tissue transplant, is also administered a therapeuticallyeffective amount of TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology. In some embodiments, the composition is administered to thesubject prior to organ or tissue transplant, during organ or tissuetransplant and/or after an organ or tissue transplant. In someembodiments, the subject would receive a combination of (i) TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or (ii) peptideconjugates of the present technology and cyclosporine before, duringand/or after an organ or tissue transplant. The composition ormedicament including TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology and optionally, cyclosporine, would be administered in anamount sufficient to cure, or partially arrest, the symptoms ofnephrotoxicity, including its complications and intermediatepathological phenotypes. For example, in some embodiments, thecompositions or medicaments are administered in an amount sufficient toeliminate the risk of, reduce the risk of, or delay the onset ofnephrotoxicity, including biochemical, histologic and/or behavioralsymptoms of the condition, its complications and intermediatepathological phenotypes. Administration of prophylactic TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology and cyclosporine can occur prior tothe manifestation of symptoms characteristic of the aberrancy, such thatthe condition is prevented or, alternatively, delayed in itsprogression. Typically, subjects who receive the composition will have ahealthier transplanted organ or tissue, and/or are able to maintain ahigher and/or more consistent cyclosporine dosage or regimen for longerperiods of time compared to subjects who do not receive the composition.In some embodiments, patients receiving TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) alone or in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology or pharmaceutically acceptable salt thereof such as anacetate, tartrate, or trifluoroacetate salt, in conjunction withcyclosporine are able to tolerate longer and/or more consistentcyclosporine treatment regimens, and/or higher doses of cyclosporine. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, patients receiving TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology or a pharmaceutically acceptablesalt thereof such as an acetate, tartrate, or trifluoroacetate salt, inconjunction with cyclosporine, will have an increased tolerance forcyclosporine as compared to a patient who is not receiving thecomposition. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in decreasing islet cell apoptosis andenhancing viability of islet cells after transplantation. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology described herein are useful in reducing oxidativedamage in a mammal in need thereof. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. Mammals in need of reducing oxidativedamage are those mammals suffering from a disease, condition ortreatment associated with oxidative damage. Typically, oxidative damageis caused by free radicals, such as reactive oxygen species (ROS) and/orreactive nitrogen species (RNS). Examples of ROS and RNS includehydroxyl radical, superoxide anion radical, nitric oxide, hydrogen,hypochlorous acid (HOCl) and peroxynitrite anion. Oxidative damage isconsidered to be “reduced” if the amount of oxidative damage in amammal, a removed organ, or a cell is decreased after administration ofan effective amount of the TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology.

In some embodiments, a mammal to be treated can be a mammal with adisease or condition associated with oxidative damage. The oxidativedamage can occur in any cell, tissue or organ of the mammal. In humans,oxidative stress is involved in many diseases. Examples includeatherosclerosis, Parkinson's disease, heart failure, myocardialinfarction, Alzheimer's disease, schizophrenia, bipolar disorder,fragile X syndrome, and chronic fatigue syndrome.

In one embodiment, a mammal may be undergoing a treatment associatedwith oxidative damage. For example, the mammal may be undergoingreperfusion. Reperfusion refers to the restoration of blood flow to anyorgan or tissue in which the flow of blood is decreased or blocked. Therestoration of blood flow during reperfusion leads to respiratory burstand formation of free radicals.

In one embodiment, the mammal may have decreased or blocked blood flowdue to hypoxia or ischemia. The loss or severe reduction in blood supplyduring hypoxia or ischemia may, for example, be due to thromboembolicstroke, coronary atherosclerosis, or peripheral vascular disease.Numerous organs and tissues are subject to ischemia or hypoxia. Examplesof such organs include brain, heart, kidney, intestine and prostate. Thetissue affected is typically muscle, such as cardiac, skeletal, orsmooth muscle. For instance, cardiac muscle ischemia or hypoxia iscommonly caused by atherosclerotic or thrombotic blockages which lead tothe reduction or loss of oxygen delivery to the cardiac tissues by thecardiac arterial and capillary blood supply. Such cardiac ischemia orhypoxia may cause pain and necrosis of the affected cardiac muscle, andultimately may lead to cardiac failure.

The methods can also be used in reducing oxidative damage associatedwith any neurodegenerative disease or condition. The neurodegenerativedisease can affect any cell, tissue or organ of the central andperipheral nervous system. Examples of such cells, tissues and organsinclude, the brain, spinal cord, neurons, ganglia, Schwann cells,astrocytes, oligodendrocytes, and microglia. The neurodegenerativecondition can be an acute condition, such as a stroke or a traumaticbrain or spinal cord injury. In another embodiment, theneurodegenerative disease or condition can be a chronicneurodegenerative condition. In a chronic neurodegenerative condition,the free radicals can, for example, cause damage to a protein. Anexample of such a protein is amyloid precursor protein. Examples ofchronic neurodegenerative diseases associated with damage by freeradicals include Parkinson's disease, Alzheimer's disease, Huntington'sdisease and Amyotrophic Lateral Sclerosis (ALS). Other conditions whichcan be treated include preeclampsia, diabetes, and symptoms of andconditions associated with aging, such as macular degeneration,wrinkles.

In one aspect, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology described herein are useful in treating any disease orcondition that is associated with mitochondria permeabilitytransitioning (MPT). In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Such diseases and conditions include, but are not limited to,ischemia and/or reperfusion of a tissue or organ, hypoxia and any of anumber of neurodegenerative diseases. Mammals in need of inhibiting orpreventing of MPT are those mammals suffering from these diseases orconditions.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in the treatment or prophylaxis ofneurodegenerative diseases associated with MPT. In other embodiments,TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. Neurodegenerative diseases associatedwith MPT include, for example, Parkinson's disease, Alzheimer's disease,Huntington's disease and Amyotrophic Lateral Sclerosis (ALS). Thecompositions disclosed herein can be used to delay the onset or slow theprogression of these and other neurodegenerative diseases associatedwith MPT. In certain embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are particularly useful in the treatment of humanssuffering from the early stages of neurodegenerative diseases associatedwith MPT and in humans predisposed to these diseases. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

Accordingly, the present disclosure describes methods and compositionsincluding TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) or peptide conjugates of the presenttechnology that are capable of reducing mitochondrial ROS production inthe diaphragm during prolonged MV, or in other skeletal muscles, e.g.,soleus or plantaris muscle, during limb immobilization, or muscle disusein general. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful as therapeutic and/or prophylactic agentsin subjects suffering from, or at risk of suffering from muscleinfirmities such as weakness, atrophy, dysfunction, etc. caused bymitochondrial derived ROS. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology decrease mitochondrial ROS production in muscle. Inother embodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. Additionally or alternatively, in some embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) orpeptide conjugates of the present technology will selectivelyconcentrate in the mitochondria of skeletal muscle and provide radicalscavenging of H₂O₂, OH—, and ONOO—, and in some embodiments, radicalscavenging occurs on a dose-dependent basis. In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods for treating muscle infirmities(e.g., weakness, atrophy, dysfunction, etc.). In other embodiments, TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In such therapeutic applications,compositions or medicaments including TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) alone or in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology or a pharmaceutically acceptable salt thereof, such asacetate, tartrate, or trifluoroacetate salt, can be administered to asubject suspected of, or already suffering from, muscle infirmity, in anamount sufficient to prevent, reduce, alleviate, or partially arrest,the symptoms of muscle infirmity, including its complications andintermediate pathological phenotypes in development of the infirmity. Assuch, the present technology provides methods of treating an individualafflicted, or suspected of suffering from muscle infirmities describedherein by administering TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology or a pharmaceutically acceptable salt thereof, such asacetate, tartrate, or trifluoroacetate salt.

In another aspect, the disclosure provides methods for preventing, orreducing the likelihood of muscle infirmity, as described herein, byadministering to the subject TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology that prevent or reduce the likelihood of the initiation orprogression of the infirmity. Subjects at risk for developing muscleinfirmity can be readily identified, e.g., a subject preparing for orabout to undergo MV or related diaphragmatic muscles disuse or any otherskeletal muscle disuse that may be envisaged by a medical professional(e.g., casting a limb).

In prophylactic applications, a pharmaceutical composition or medicamentcomprising one or more TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology or a pharmaceutically acceptable salt thereof, such asacetate, tartrate, or trifluoroacetate salt, are administered to asubject susceptible to, or otherwise at risk of muscle infirmity in anamount sufficient to eliminate or reduce the risk, or delay the onset ofmuscle infirmity, including biochemical, histologic and/or behavioralsymptoms of the infirmity, its complications and intermediatepathological phenotypes presenting during development of the infirmity.Administration of one or more TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that the disorder is prevented or,alternatively, delayed in its progression.

In some embodiments, subjects in need of protection from or treatment ofmuscle infirmity also include subjects suffering from a disease,condition or treatment associated with oxidative damage. Typically, theoxidative damage is caused by free radicals, such as reactive oxygenspecies (ROS) and/or reactive nitrogen species (RNS). Examples of ROSand RNS include hydroxyl radical (HO.), superoxide anion radical (O₂.⁻),nitric oxide (NO.), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl),and peroxynitrite anion (ONOO⁻).

A composition comprising TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to treat or prevent muscle infirmity associated with muscleimmobilization e.g., due to casting or other disuse, can be administeredat any time before, during or after the immobilization or disuse. Forexample, in some embodiments, one or more doses of a compositioncomprising TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) alone or in combination with one or moreactive agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology can be administered before muscle immobilization or disuse,immediately after muscle immobilization or disuse, during the course ofmuscle immobilization or disuse, and/or after muscle immobilization ordisuse (e.g., after cast removal). By way of example, and not by way oflimitation, in some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology can be administered once per day, twice per day, three timesper day, four times per day six times per day or more, for the durationof the immobilization or disuse. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates of the present technology can be administered daily, everyother day, twice, three times, or for times per week, or once, twicethree, four, five or six times per month for the duration of theimmobilization or disuse.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods of treating or preventingmuscle infirmity due to muscle disuse or disuse atrophy, associated withloss of muscle mass and strength. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. Atrophy is a physiological processrelating to the reabsorption and degradation of tissues, e.g., fibrousmuscle tissue, which involves apoptosis at the cellular level. Whenatrophy occurs from loss of trophic support or other disease, it isknown as pathological atrophy. Such atrophy or pathological atrophy mayresult from, or is related to, limb immobilization, prolonged limbimmobilization, casting limb immobilization, mechanical ventilation(MV), prolonged MV, extended bed rest cachexia, congestive heartfailure, liver disease, sarcopenia, wasting, poor nourishment, poorcirculation, hormonal irregularities, loss of nerve function, and thelike. Accordingly, the present methods relate to the prevention and/ortreatment of muscle infirmities in a subject, including skeletal muscleatrophy, comprising administering an effective amount of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates to a subject in need thereof.

Additional examples of muscle infirmities which can be treated,prevented, or alleviated by administering the compositions andformulations disclosed herein include, without limitation, age-relatedmuscle infirmities, muscle infirmities associated with prolonged bedrest, muscle infirmities such as weakness and atrophy associated withmicrogravity, as in space flight, muscle infirmities associated witheffects of certain drugs (e.g., statins, antiretrovirals, andthiazolidinediones (TZDs), and muscle infirmities such as cachexia, forexample cachexia caused by cancer or other diseases.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in the treatment or prevention of ananatomic zone of no re-flow to a subject in need thereof. In otherembodiments, TBMs (or derivatives, analogues, or pharmaceuticallyacceptable salts thereof) in combination with one or more active agents(e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In one embodiment, the administration of TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) alone or incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptideconjugates to a subject is done before the formation of the anatomiczone of no re-flow. In another embodiment, the administration of TBMs(or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology to a subject is done after the formation of an anatomic zoneof no re-flow. In one embodiment, the method is performed in conjunctionwith a revascularization procedure. Also provided is a method for thetreatment or prevention of cardiac ischemia-reperfusion injury. Alsoprovided is a method of treating a myocardial infarction in a subject toprevent injury to the heart upon reperfusion. In one aspect, the presenttechnology relates to a method of coronary revascularization comprisingadministering to a mammalian subject a therapeutically effective amountof TBMs (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) alone or in combination with one or more active agents (e.g.,an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology and performing a coronary artery bypass graft (CABG)procedure on the subject.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods of preventing an anatomic zoneof no re-flow in a subject, which prevent the initiation or progressionof the condition. In other embodiments, TBMs (or derivatives, analogues,or pharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

Subjects at risk for an anatomic zone of no re-flow can be identifiedby, e.g., any or a combination of diagnostic or prognostic assays asdescribed herein. In prophylactic applications, pharmaceuticalcompositions or medicaments of TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject susceptible to, or otherwise atrisk of a disease or condition in an amount sufficient to eliminate orreduce the risk, or delay the onset of the disease or condition,including biochemical, histologic and/or behavioral symptoms of thedisease or condition, its complications and intermediate pathologicalphenotypes presenting during development of the disease or condition.Administration of a prophylactic TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) alone or in combination withone or more active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

In some embodiments, the aromatic-cationic peptide, TBM, or peptideconjugate of the present technology is administered to a subject in anamount effective to protect the subject from acute renal injury (ARI) oracute liver failure (ALF). Also, the aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology may be administered to asubject in an amount effective in treating ARI or ALF.

As used herein, the term “effective amount” or “pharmaceuticallyeffective amount” or “therapeutically effective amount” of acomposition, is a quantity sufficient to achieve a desired therapeuticand/or prophylactic effect, e.g., an amount which results in theprevention of, or a decrease in, the symptoms associated with ARI orALF. The amount of a composition of the present technology administeredto the subject will depend on the type and severity of the disease andon the characteristics of the individual, such as general health, age,sex, body weight and tolerance to drugs. It will also depend on thedegree, severity and type of disease. The skilled artisan will be ableto determine appropriate dosages depending on these and other factors.The compositions of the present technology can also be administered incombination with one or more additional therapeutic compounds. In thepresent methods, aromatic-cationic peptide, TBM, or peptide conjugate ofthe present technology may be administered to a subject having one ormore signs of ARI caused by a disease or condition. Administration of aneffective amount of the aromatic-cationic peptide, TBM, or peptideconjugate of the present technology may improve at least one sign orsymptom of ARI in the subject, e.g., metabolic acidosis (acidificationof the blood), hyperkalemia (elevated potassium levels), oliguria, oranuria (decrease or cessation of urine production), changes in bodyfluid balance, and effects on other organ systems. For example, a“therapeutically effective amount” of the aromatic-cationic peptide,TBM, or peptide conjugate of the present technology means a level atwhich the physiological effects of acute renal failure will be kept at aminimum. Typically, the efficacy of the biological effect is measured incomparison to a subject or class of subjects not administered thecompounds.

Any method known to those in the art for contacting a cell, organ ortissue with an aromatic-cationic peptide, TBM, or peptide conjugate ofthe present technology may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of aromatic-cationic peptides, TBMs, or peptideconjugates of the present technology, such as those described herein, toa mammal, such as a human. When used in vivo for therapy, anaromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology is administered to the subject in effective amounts (i.e.,amounts that have desired therapeutic effect). Compositions willnormally be administered parenteral, topically, or orally. The dose anddosage regimen will depend upon the type and severity of disease orinjury, the characteristics of the particular aromatic-cationic peptide,TBM, or peptide conjugate of the present technology e.g., itstherapeutic index, the characteristics of the subject, and the subject'smedical history.

In some embodiments, the dosage of the aromatic-cationic peptide, TBM,or peptide conjugate of the present technology is provided at a “low,”“mid,” or “high” dose level. In some embodiments, the low dose is fromabout 0.001 to about 0.5 mg/kg/h, or from about 0.01 to about 0.1mg/kg/h. In some embodiments, the mid-dose is from about 0.1 to about1.0 mg/kg/h, or from about 0.1 to about 0.5 mg/kg/h. In someembodiments, the high dose is from about 0.5 to about 10 mg/kg/h, orfrom about 0.5 to about 2 mg/kg/h. The skilled artisan will appreciatethat certain factors may influence the dosage and timing required toeffectively treat a subject, including but not limited to, the severityof the medical disease or condition, previous treatments, the generalhealth and/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of thearomatic-cationic peptides, TBMs, or peptide conjugates described hereincan include a single treatment or a series of treatments.

In some embodiments, the aromatic-cationic peptide, TBM, or peptideconjugate of the present technology is administered in combination withanother therapeutic agent. By way of example, a patient receiving anaromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology who experiences inflammation may be co-administered ananti-inflammatory agent. By way of example, the therapeuticeffectiveness of the aromatic-cationic peptide, TBM, or peptideconjugate of the present technology may be enhanced by co-administrationof an adjuvant. By way of example, the therapeutic benefit to a patientmay be increased by administering an aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology in combination with anothertherapeutic agent known or suspected to aid in the prevention ortreatment of a particular condition.

Non-limiting examples of combination therapies include use of one ormore aromatic-cationic peptides, TBMs, or peptide conjugates of thepresent technology together with nitric oxide (NO) inducers, statins,negatively charged phospholipids, antioxidants, minerals,anti-inflammatory agents, anti-angiogenic agents, matrixmetalloproteinase inhibitors, or carotenoids. In some embodiments,agents used in combination with compositions described herein may fallwithin multiple categories (for example, lutein is both an antioxidantand a carotenoid). Further, the aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology may be administered withadditional agents that may provide benefit to the patient, including byway of example only cyclosporin A.

In addition, the aromatic-cationic peptide, TBM, or peptide conjugate ofthe present technology may also be used in combination with proceduresthat may provide additional or synergistic benefit to the patient,including, for example, extracorporeal rheopheresis (membranedifferential filtration), implantable miniature telescopes, laserphotocoagulation of drusen, and microstimulation therapy.

The use of antioxidants has been shown to benefit patients with maculardegenerations and dystrophies. See, e.g., Arch. Ophthalmol. 119:1417-36(2001); Sparrow, et al., J. Biol. Chem. 278:18207-13 (2003).Non-limiting examples of antioxidants suitable for use in combinationwith at least one aromatic-cationic peptide, TBM, or peptide conjugateof the present technology include vitamin C, vitamin E, beta-caroteneand other carotenoids, coenzyme Q,4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), lutein,butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E),and bilberry extract.

The use of certain minerals has also been shown to benefit patients withmacular degenerations and dystrophies. See, e.g., Arch. Ophthalmol.,119:1417-36 (2001). Non-limiting examples of minerals for use incombination with at least one aromatic-cationic peptide, TBM, or peptideconjugate of the present technology include copper-containing minerals(e.g., cupric oxide), zinc-containing minerals (e.g., zinc oxide), andselenium-containing compounds.

The use of certain negatively-charged phospholipids has also been shownto benefit patients with macular degenerations and dystrophies. See,e.g., Shaban & Richter, Biol., Chem. 383:537-45 (2002); Shaban, et al.,Exp. Eye Res. 75:99-108 (2002). Non-limiting examples of negativelycharged phospholipids suitable for use in combination with at least onearomatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology include cardiolipin and phosphatidylglycerol.Positively-charged and/or neutral phospholipids may also provide benefitfor patients with macular degenerations and dystrophies when used incombination with aromatic-cationic peptide, TBM, or peptide conjugate ofthe present technology.

The use of certain carotenoids has been correlated with the maintenanceof photoprotection necessary in photoreceptor cells. Carotenoids arenaturally-occurring yellow to red pigments of the terpenoid group thatcan be found in plants, algae, bacteria, and certain animals, such asbirds and shellfish. Carotenoids are a large class of molecules in whichmore than 600 naturally occurring species have been identified.Carotenoids include hydrocarbons (carotenes) and their oxygenated,alcoholic derivatives (xanthophylls). They include actinioerythrol,astaxanthin, canthaxanthin, capsanthin, capsorubin, β-8′-apocarotenal(apo-carotenal), β-12′-apo-carotenal, α-carotene, β-carotene, “carotene”(a mixture of α- and β-carotenes), γ-carotenes, β-cryptoxanthin, lutein,lycopene, violerythrin, zeaxanthin, and esters of hydroxyl- orcarboxyl-containing members. Many of the carotenoids occur in nature ascis- and trans-isomeric forms, while synthetic compounds frequentlyexist as racemic mixtures.

In humans, the retina selectively accumulates mainly two carotenoids:zeaxanthin and lutein. These two carotenoids are thought to aid inprotecting the retina because they are powerful antioxidants and absorbblue light. Studies with quails have established that animals raised oncarotenoid-deficient diets develop retinas with low concentrations ofzeaxanthin and suffer severe light damage, as evidenced by a very highnumber of apoptotic photoreceptor cells. By contrast, animals raised onhigh-carotenoid diets develop retinas with high zeaxanthinconcentrations that sustain minimal light damage. Non-limiting examplesof carotenoids suitable for use in combination with at least onearomatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology include lutein and zeaxanthin, as well as any of theaforementioned carotenoids.

Nitric oxide inducers include compounds that stimulate endogenous NO orelevate levels of endogenous endothelium-derived relaxing factor (EDRF)in vivo, or are substrates for nitric oxide synthase. Such compoundsinclude, for example, L-arginine, L-homoarginine, andN-hydroxy-L-arginine, including their nitrosated and nitrosylatedanalogues (e.g., nitrosated L-arginine, nitrosylated L-arginine,nitrosated N-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine,nitrosated L-homoarginine and nitrosylated L-homoarginine), precursorsof L-arginine and/or physiologically acceptable salts thereof,including, for example, citrulline, ornithine, glutamine, lysine,polypeptides comprising at least one of these amino acids, inhibitors ofthe enzyme arginase (e.g., N-hydroxy-L-arginine and2(S)-amino-6-boronohexanoic acid) and the substrates for nitric oxidesynthase, cytokines, adenosine, bradykinin, calreticulin, bisacodyl, andphenolphthalein. EDRF is a vascular relaxing factor secreted by theendothelium, and has been identified as nitric oxide or a closelyrelated derivative thereof (Palmer, et al., Nature 327:524-526 (1987);Ignarro, et al., Proc. Natl. Acad. Sci. 84:9265-9269 (1987)). In someembodiments, the aromatic-cationic peptides, TBMs, or peptide conjugatesof the present technology may also be used in combination with NOinducers.

Statins serve as lipid-lowering agents and/or suitable nitric oxideinducers. In addition, a relationship has been demonstrated betweenstatin use and delayed onset or development of macular degeneration. G.McGwin, et al., Br. J. Ophthalmol. 87:1121-25 (2003). Statins can thusprovide benefit to a patient suffering from an ophthalmic condition(such as the macular degenerations and dystrophies, and the retinaldystrophies) when administered in combination with aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology. Suitablestatins include, by way of example only, rosuvastatin, pitivastatin,simvastatin, pravastatin, cerivastatin, mevastatin, vicrostatin,fluvastatin, compactin, lovastatin, dalvastatin, fluindostatin,atorvastatin, atorvastatin calcium (which is the hemicalcium salt ofatorvastatin), and dihydrocompactin.

Suitable anti-inflammatory agents for use in combination with thearomatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may also be used in combination with include, by way ofexample only, aspirin and other salicylates, cromolyn, nedocromil,theophylline, zileuton, zafirlukast, montelukast, pranlukast,indomethacin, lipoxygenase inhibitors, non-steroidal anti-inflammatorydrugs (NSAIDs) (e.g., ibuprofen and naproxin), prednisone,dexamethasone, cyclooxygenase inhibitors (i.e., COX-1 and/or COX-2inhibitors such as NAPROXEN™, and CELEBREX™), statins (e.g.,rosuvastatin, pitivastatin, simvastatin, pravastatin, cerivastatin,mevastatin, velostatin, fluvastatin, compactin, lovastatin, dalvastatin,fluindostatin, atorvastatin, atorvastatin calcium (hemicalcium salt ofatorvastatin), dihydrocompactin), and disassociated steroids.

Matrix metalloproteinase (MMP) inhibitors may also be administered incombination with compositions described herein for the treatment ofophthalmic conditions or symptoms associated with macular or retinaldegeneration. MMPs are known to hydrolyze most components of theextracellular matrix. These proteinases play a central role in manybiological processes such as normal tissue remodeling, embryogenesis,wound healing, and angiogenesis. However, high levels of MMPs areassociated with many disease states, including macular degeneration.Many MMPs have been identified, most of which are multi-domain zincendopeptidases. A number of metalloproteinase inhibitors are known (see,e.g., Whittaker, et al., Chem. Rev. 99(9):2735-2776 (1999)).Representative examples of MMP inhibitors include tissue inhibitors ofmetalloproteinases (TIMPs) (e.g., TIMP-1, TIMP-2, TIMP-3, TIMP-4),α-2-macroglobulin, tetracyclines (e.g., tetracycline, minocycline,doxycycline), hydroxamates (e.g., BATIMASTAT™, MARIMISTAT™ andTROCADE™), chelators (e.g., EDTA, cysteine, acetylcysteine,D-penicillamine, gold salts), synthetic MMP fragments, succinylmercaptopurines, phosphonamidates, and hydroxaminic acids. Non-limitingexamples of MMP inhibitors suitable for use in combination withcompositions described herein include any of the aforementionedinhibitors.

The use of anti-angiogenic or anti-VEGF drugs has also been shown toprovide benefit for patients with macular degenerations and dystrophies.Examples of suitable anti-angiogenic or anti-VEGF drugs for use incombination with at least one aromatic-cationic peptide, TBM, or peptideconjugate of the present technology may also be used in combination withinclude rhufab V2 (LUCCNTIS™), Tryptophanyl-tRNA synthetase (TrpRS),eye001 (anti-VEGF pegylated aptamer), squalamine, RETAANE™ (anecortaveacetate for depot suspension), combretastatin A4 prodrug (CA4P),MACUGEN™ MIFEPREX™ (mifepristone-ru486), subtenon triamcinoloneacetonide, intravitreal crystalline triamcinolone acetonide, prinomastat(AG3340), fluocinolone acetonide (including fluocinolone intraocularimplant), VEGFR inhibitors, and VEGF-Trap.

Other pharmaceutical therapies that have been used to relieve visualimpairment can be used in combination with at least onearomatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may also be used in combination with. Such treatments includebut are not limited to agents such as VISUDYNC™ with use of anon-thermal laser, PKC 412, endovion, neurotrophic factors (e.g., glialderived neurotrophic factor, ciliary neurotrophic factor), diatazem,dorzolamide, phototrop, 9-cis-retinal, eye medication (including EchoTherapy) including phospholine iodide or echothiophate or carbonicanhydrase inhibitors, AE-941, Sima-027, pegaptanib, neurotrophins (e.g.,NT-4/5), cand5, ranibizumab, INS-37217, integrin antagonists, EG-3306,BDM-E, thalidomide, cardiotrophin-1, 2-methoxyestradiol, DL8234,NTC-200, tetrathiomolybdate, LYN-002, microalgal compound, D-9120,ATX-S10, TGF-beta 2, tyrosine kinase inhibitors, NX-278-L, Opt-24,retinal cell ganglion neuroprotectants, N-nitropyrazole derivatives,KP-I02, and cyclosporin A.

Multiple therapeutic agents may be administered in any order orsimultaneously. If simultaneously, the agents may be provided in asingle, unified form, or in multiple forms (i.e. as a single solution oras two separate solutions). One of the therapeutic agents may be givenin multiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than about four weeks, less than about sixweeks, less than about 2 months, less than about 4 months, less thanabout 6 months, or less than about one year. In addition, thecombination methods, compositions, and formulations are not limited tothe use of only two agents. By way of example, an aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology may beprovided with at least one antioxidant and at least one negativelycharged phospholipid. By way of example, an aromatic-cationic peptide,TBM, or peptide conjugate of the present technology may be provided withat least one antioxidant and at least one inducer of nitric oxideproduction. By way of example, an aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology may be provided with atleast one inducer of nitric oxide productions and at least onenegatively charged phospholipid.

In addition, an aromatic-cationic peptide, TBM, or peptide conjugate ofthe present technology may be used in combination with procedures thatmay provide additional or synergistic benefits to the patient. Forexample, procedures known, proposed, or considered to relieve visualimpairment include but are not limited to “limited retinaltranslocation,” photodynamic therapy (e.g., receptor-targeted PDT,porfimer sodium for injection with PDT, verteporfin, rostaporfin withPDT, talaporfin sodium with PDT, motexafin lutetium), antisenseoligonucleotides (e.g., products of Novagali Pharma SA, ISIS-13650),laser photocoagulation, drusen lasering, macular hole surgery, maculartranslocation surgery, implantable miniature telescopes, phi-motionangiography (micro-laser therapy and feeder vessel treatment), protonbeam therapy, microstimulation therapy, retinal detachment and vitreoussurgery, scleral buckle, submacular surgery, transpupillarythermotherapy, photosystem I therapy, use of RNA interference (RNAi),extracorporeal rheopheresis (membrane differential filtration andrheotherapy), microchip implantation, stem cell therapy, genereplacement therapy, ribozyme gene therapy (including gene therapy forhypoxia response element, LENTIPAC™, PDEF gene therapy),photoreceptor/retinal cell transplantation (including transplantableretinal epithelial cells, retinal cell transplant), and acupuncture.

Further combinations that may be used to benefit an individual includeusing genetic testing to determine whether that individual is a carrierof a mutant gene that is known to be correlated with certain ophthalmicconditions. By way of example only, defects in the human ABCA4 gene arethought to be associated with five distinct retinal phenotypes includingStargardt disease, cone-rod dystrophy, age-related macular degenerationand retinitis pigmentosa. See e.g., Allikmets, et al., Science277:1805-07 (1997); Lewis, et al., Am. Hum. Genet. 64:422-34 (1999);Stone, et al., Nature Genetics 20:328-29 (1998); Allikmets, Am. J Hum.Gen. 67:793-799 (2000); Klevering, et al., Ophthalmology 11 1:546-553(2004). In addition, an autosomal dominant form of Stargardt Disease iscaused by mutations in the ELOV4 gene. See Karan, et al., Proc. Natl.Acad. Sci. (2005). Patients possessing any of these mutations areexpected to benefit from the therapeutic and/or prophylactic methodsdescribed herein.

In some embodiments, aromatic-cationic peptides, TBMs, or peptideconjugates of the present technology are combined with one or moreadditional agents for the prevention or treatment of heart failure. Drugtreatment for heart failure typically involves diuretics,angiotensin-converting-enzyme (ACE) inhibitors, digoxin (digitalis),calcium channel blockers, and beta-blockers. In mild cases, thiazidediuretics, such as hydrochlorothiazide at 25-50 mg/day or chlorothiazideat 250-500 mg/day, are useful. However, supplemental potassium chloridemay be needed, since chronic diuresis causes hypokalemis alkalosis.Moreover, thiazide diuretics usually are not effective in patients withadvanced symptoms of heart failure. Typical doses of ACE inhibitorsinclude captopril at 2550 mg/day and quinapril at 10 mg/day.

In one embodiment, an aromatic-cationic peptide, TBM, or peptideconjugate of the present technology is combined with an adrenergicbeta-2 agonist. An “adrenergic beta-2 agonist” refers to adrenergicbeta-2 agonists and analogues and derivatives thereof, including, forexample, natural or synthetic functional variants which have adrenergicbeta-2 agonist biological activity, as well as fragments of anadrenergic beta-2 agonist having adrenergic beta-2 agonist biologicalactivity. The term “adrenergic beta-2 agonist biological activity”refers to activity that mimics the effects of adrenaline andnoradrenaline in a subject and which improves myocardial contractilityin a patient having heart failure. Commonly known adrenergic beta-2agonists include, but are not limited to, clenbuterol, albuterol,formeoterol, levalbuterol, metaproterenol, pirbuterol, salmeterol, andterbutaline.

In one embodiment, an aromatic-cationic peptide, TBM, or peptideconjugate of the present technology is combined with an adrenergicbeta-1 antagonist. Adrenergic beta-1 antagonists and adrenergic beta-1blockers refer to adrenergic beta-1 antagonists and analogues andderivatives thereof, including, for example, natural or syntheticfunctional variants which have adrenergic beta-1 antagonist biologicalactivity, as well as fragments of an adrenergic beta-1 antagonist havingadrenergic beta-1 antagonist biological activity. Adrenergic beta-1antagonist biological activity refers to activity that blocks theeffects of adrenaline on beta receptors. Commonly known adrenergicbeta-1 antagonists include, but are not limited to, acebutolol,atenolol, betaxolol, bisoprolol, esmolol, and metoprolol.

Clenbuterol, for example, is available under numerous brand namesincluding Spiropent, BRONCODIL®, BRONEOTEROL®, Cesbron, and Clenbuter.Similarly, methods of preparing adrenergic beta-1 antagonists such asmetoprolol and their analogues and derivatives are well-known in theart. Metoprolol, in particular, is commercially available under thebrand names LOPRESSOR® (metoprolol tartate) manufactured by NovartisPharmaceuticals Corporation (East Hanover, N.J., USA). Generic versionsof LOPRESSOR® are also available from Mylan Laboratories Inc.(Canonsburg, Pa., USA); and Watson Pharmaceuticals, Inc. (Morristown,N.J., USA). Metoprolol is also commercially available under the brandname Toprol XL®, manufactured by Astra Zeneca, LP (London, G.B.).

In one embodiment, an additional therapeutic agent is administered to asubject in combination with an aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology, such that a synergistictherapeutic effect is produced.

In one embodiment, the subject is administered a composition describedherein prior to ischemia. In one embodiment, the subject is administeredthe composition prior to the reperfusion of ischemic tissue. In oneembodiment, the subject is administered the composition at about thetime of reperfusion of ischemic tissue. In one embodiment, the subjectis administered the composition after reperfusion of ischemic tissue.

In one embodiment, the subject is administered a composition describedherein prior to the CABG or revascularization procedure. In anotherembodiment, the subject is administered the composition after the CABGor revascularization procedure. In another embodiment, the subject isadministered the composition during and after the CABG orrevascularization procedure. In another embodiment, the subject isadministered the composition continuously before, during, and after theCABG or revascularization procedure.

In one embodiment, the subject is administered a composition describedherein starting at least 5 minutes, at least 10 minutes, at least 30minutes, at least 1 hour, at least 3 hours, at least 5 hours, at least 8hours, at least 12 hours, or at least 24 hours prior to CABG orrevascularization, i.e., reperfusion of ischemic tissue. In oneembodiment, the subject is administered the composition from about 5-30minutes, from about 10-60 minutes, from about 10-90 minutes, or fromabout 10-120 minutes prior to the CABG or revascularization procedure.In one embodiment, the subject is administered the composition untilabout 5-30 minutes, until about 10-60 minutes, until about 10-90minutes, until about 10-120 minutes, or until about 10-180 minutes afterthe CABG or revascularization procedure.

In one embodiment, the subject is administered the composition for atleast 30 min, at least 1 hour, at least 3 hours, at least 5 hours, atleast 8 hours, at least 12 hours, or at least 24 hours after the CABGprocedure or revascularization procedure, i.e., reperfusion of ischemictissue. In one embodiment, the composition is administered until about30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 8 hours, about 12 hours, or about 24 hours afterthe CABG procedure or revascularization procedure i.e., reperfusion ofischemic tissue.

In one embodiment, the subject is administered the composition as an IVinfusion starting at about 1 minute to 30 minutes prior to reperfusion(i.e. about 5 minutes, about 10 minutes, about 20 minutes, or about 30minutes prior to reperfusion) and continuing for about 1 hour to about24 hours after reperfusion (i.e., about 1 hour, about 2 hours, about 3hours, about 4 hours, etc. after reperfusion). In one embodiment, thesubject receives an IV bolus injection prior to reperfusion of thetissue. In one embodiment, the subject continues to receive thecomposition chronically after the reperfusion period, i.e., for about1-7 days, about 1-14 days, or about 1-30 days after the reperfusionperiod. During this period, the composition may be administered by anyroute, e.g., subcutaneously or intravenously.

In one embodiment, the composition is administered by a systemicintravenous infusion commencing about 5-60 minutes, about 10-45 minutes,or about 30 minutes before the induction of anesthesia. In oneembodiment, the composition is administered in conjunction with acardioplegic solution. In one embodiment, the composition isadministered as part of the priming solution in a heart lung machineduring cardiopulmonary bypass.

In various embodiments, the subject is suffering from a myocardialinfarction, a stroke, or is in need of angioplasty. In one embodiment, arevascularization procedure is selected from the group consisting ofballoon angioplasty, insertion of a stent, percutaneous coronaryintervention (PCI), percutaneous transluminal coronary angioplasty, ordirectional coronary atherectomy. In one embodiment, therevascularization procedure comprises the removal of the occlusion. Inone embodiment, the revascularization procedure comprises theadministration of one or more thrombolytic agents. In one embodiment,the one or more thrombolytic agents is selected from the groupconsisting of: tissue plasminogen activator, urokinase, prourokinase,streptokinase, acylated form of plasminogen, acylated form of plasmin,and acylated streptokinase-plasminogen complex.

In one embodiment the vessel occlusion comprises a cardiac vesselocclusion. In another embodiment, the vessel occlusion is anintracranial vessel occlusion. In yet other embodiments, the vesselocclusion is selected from the group consisting of: deep venousthrombosis; peripheral thrombosis; embolic thrombosis; hepatic veinthrombosis; sinus thrombosis: venous thrombosis; an occludedarterio-venal shunt; and an occluded catheter device.

In one aspect, the present technology relates to the treatment ofatherosclerotic vascular disease (ARVD) comprising administering to asubject in need thereof therapeutically effective amounts ofaromatic-cationic peptides, TBMs, or peptide conjugates of the presenttechnology. In some embodiments, the treatment is chronic treatment,administered for a period of greater than 1 week.

In another aspect, the present technology relates to the treatment orprevention of ischemic injury in the absence of tissue reperfusion. Forexample, compositions may be administered to patients experiencing acuteischemia in one or more tissues or organs who, for example, are notsuitable candidates for revascularization procedures or for whomrevascularization procedures are not readily available. Additionally oralternatively, the compositions may be administered to patients withchronic ischemia in one or more tissues in order to forestall the needfor a revascularization procedure. Patients administered compositionsfor the treatment or prevention of ischemic injury in the absence oftissue reperfusion may additionally be administered compositions priorto, during, and subsequent to revascularization procedures according tothe methods described herein.

In one embodiment, the treatment of renal reperfusion injury includesincreasing the amount or area of tissue perfusion in a subject comparedto a similar subject not administered the composition. In oneembodiment, the prevention of renal reperfusion injury includes reducingthe amount or area of microvascular damage caused by reperfusion in asubject compared to a similar subject not administered the composition.In some embodiments, treatment or prevention of renal reperfusion injuryincludes reducing injury to the affected vessel upon reperfusion,reducing the effect of plugging by blood cells, and/or reducingendothelial cell swelling in a subject compared to a similar subject notadministered the composition. The extent of the prevention or treatmentcan be measured by any technique known in the art, including but notlimited to measurement of renal volume, renal arterial pressure, renalblood flow (RBF), and glomerular filtration rate (GFR), as well as byimaging techniques known in the art, including, but not limited to CTand micro-CT. Successful prevention or treatment can be determined bycomparing the extent of renal reperfusion injury in the subject observedby any of these imaging techniques compared to a control subject or apopulation of control subjects that are not administered thecomposition.

In one embodiment, the administration of the composition to a subject isbefore the occurrence of renal reperfusion injury. For example, in someembodiments, the composition is administered to inhibit, prevent ortreat ischemic injury in a subject in need thereof, and/or to forestallreperfusion treatment and/or alleviate or ameliorate reperfusion injury.Additionally or alternatively, in some embodiments, the administrationof the composition to a subject is after the occurrence of renalreperfusion injury. In one embodiment, the method is performed inconjunction with a revascularization procedure. In one embodiment, therevascularization procedure is percutaneous transluminal renalangioplasty (PTRA). In one aspect, the present technology relates to amethod of renal revascularization comprising administering to amammalian subject a therapeutically effective amount of the compositionand performing PTRA on the subject.

In one embodiment, the subject is administered an aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology, prior to arevascularization procedure. In another embodiment, the subject isadministered the aromatic-cationic peptide, TBM, and/or peptideconjugate of the present technology after the revascularizationprocedure. In another embodiment, the subject is administered thearomatic-cationic peptide, TBM, and/or peptide conjugate of the presenttechnology during and after the revascularization procedure. In yetanother embodiment, the subject is administered the aromatic-cationicpeptide, TBM, and/or peptide conjugate of the present technologycontinuously before, during, and after the revascularization procedure.In another embodiment, the subject is administered the aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology regularly(i.e., chronically) following renal artery stenosis and/or a renalrevascularization procedure.

In some embodiments, the subject is administered the aromatic-cationicpeptide, TBM, and/or peptide conjugate of the present technology afterthe revascularization procedure. In one embodiment, the subject isadministered the aromatic-cationic peptide, TBM, and/or peptideconjugate of the present technology for at least 3 hours, at least 5hours, at least 8 hours, at least 12 hours, or at least 24 hours afterthe revascularization procedure. In some embodiments, the subject isadministered the aromatic-cationic peptide, TBM, and/or peptideconjugate of the present technology prior to the revascularizationprocedure. In one embodiment, the subject is administered thearomatic-cationic peptide, TBM, and/or peptide conjugate of the presenttechnology starting at least 8 hours, at least 4 hours, at least 2hours, at least 1 hour, or at least 10 minutes prior to therevascularization procedure. In one embodiment, the subject isadministered the aromatic-cationic peptide, TBM, and/or peptideconjugate of the present technology for at least one week, at least onemonth or at least one year after the revascularization procedure. Insome embodiments, the subject is administered the aromatic-cationicpeptide, TBM, and/or peptide conjugate of the present technology priorto and after the revascularization procedure. In some embodiments, thesubject is administered the aromatic-cationic peptide, TBM, and/orpeptide conjugate of the present technology as an infusion over aspecified period of time. In some embodiments, the aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology isadministered to the subject as a bolus.

In some embodiments, the present methods comprise administration ofaromatic-cationic peptide, TBM, and/or peptide conjugate of the presenttechnology in conjunction with one or more thrombolytic agents. In someembodiments, the one or more thrombolytic agents are selected from thegroup consisting of: tissue plasminogen activator, urokinase,prourokinase, streptokinase, acylated form of plasminogen, acylated formof plasmin, and acylated streptokinase-plasminogen complex.

In some embodiments, TBMs, (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful in methods of treating vessel occlusioninjury, an anatomic zone of no re-flow, or cardiac ischemia-reperfusioninjury in a subject for therapeutic purposes. In other embodiments,TBMs, (or derivatives, analogues, or pharmaceutically acceptable saltsthereof) in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In therapeutic applications,compositions or medicaments comprising TBMs, (or derivatives, analogues,or pharmaceutically acceptable salts thereof) alone or in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or peptide conjugates of the presenttechnology are administered to a subject suspected of, or alreadysuffering from such a disease or condition in an amount sufficient tocure, or partially arrest, the symptoms of the disease or condition,including its complications and intermediate pathological phenotypes indevelopment of the disease or condition. As such, the present technologyprovides methods of treating an individual afflicted with an anatomiczone of no re-flow.

Pain Management/Analgesia

In one aspect, the present disclosure provides a method for stimulatinga mu-opioid receptor in a mammal in need thereof. The method comprisesadministering systemically to the mammal an effective amount of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) or peptideconjugates of the present technology. In one embodiment, the methodcomprises inhibiting norepinephrine in the mammal.

As used herein, “neuropathy” or “peripheral neuropathy” refers generallyto damage to nerves of the peripheral nervous system. The termencompasses neuropathy of various etiologies, including but not limitedto acquired neuropathies, hereditary neuropathies, and idiopathicneuropathies. Illustrative neuropathies include but are not limited toneuropathies caused by, resulting from, or otherwise associated withtrauma, genetic disorders, metabolic/endocrine complications,inflammatory diseases, infectious diseases, vitamin deficiencies,malignant diseases, and toxicity, such as alcohol, organic metal, heavymetal, radiation, and drug toxicity. As used herein, the termencompasses motor, sensory, mixed sensorimotor, chronic, and acuteneuropathy. As used herein the term encompasses mononeuropathy, multiplemononeuropathy, and polyneuropathy.

Drug toxicity causes multiple forms of peripheral neuropathy, with themost common being axonal degeneration. A notable exception is that ofperhexiline, a prophylactic anti-anginal agent that can cause segmentaldemyelination, a localized degeneration of the insulating layer aroundsome nerves.

Peripheral neuropathies usually present sensory symptoms initially, andoften progress to motor disorders. Most drug-induced peripheralneuropathies are purely sensory or mixed sensorimotor defects. A notableexception here is that of Dapzone, which causes an almost exclusivelymotor neuropathy.

Drug-induced peripheral neuropathy, including, for example,chemotherapy-induced peripheral neuropathy can cause a variety ofdose-limiting neuropathic conditions, including 1) myalgias, 2) painfulburning paresthesis, 3) glove-and-stocking sensory neuropathy, and 4)hyperalgia and allodynia. Hyperalgia refers to hypersensitivity and paincaused by stimuli that is normally only mildly painful or irritating.Allodynia refers to hypersensitivity and pain caused by stimuli that isnormally not painful or irritating.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂)are useful for the treatment or prevention of peripheral neuropathy orthe symptoms of peripheral neuropathy. In other embodiments, TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof) incombination with one or more active agents (e.g., an aromatic-cationicpeptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show asynergistic effect in this regard. In some embodiments, the peripheralneuropathy is drug-induced peripheral neuropathy. In some embodiments,the peripheral neuropathy is induced by a chemotherapeutic agent. Insome embodiments, the chemotherapeutic agent is a vinca alkaloid. Insome embodiments, the vinca alkaloid is vincristine. In someembodiments, the symptoms of peripheral neuropathy include hyperalgesia.

As used herein, “hyperalgesia” refers to an increased sensitivity topain, which may be caused by damage to nociceptors or peripheral nerves(i.e. neuropathy). The term refers to temporary and permanenthyperalgesia, and encompasses both primary hyperalgesia (i.e. painsensitivity occurring directly in damaged tissues) and secondaryhyperalgesia (i.e. pain sensitivity occurring in undamaged tissuessurrounding damaged tissues). The term encompasses hyperalgesia causedby but not limited to neuropathy caused by, resulting from, or otherwiseassociated with genetic disorders, metabolic/endocrine complications,inflammatory diseases, vitamin deficiencies, malignant diseases, andtoxicity, such as alcohol, organic metal, heavy metal, radiation, anddrug toxicity. In some embodiments hyperalgesia is caused bydrug-induced peripheral neuropathy.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology are useful for the treatment or prevention ofhyperalgesia. In other embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) in combination with one ormore active agents (e.g., an aromatic-cationic peptide such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard. In some embodiments, the hyperalgesia is drug-induced. In someembodiments, the hyperalgesia is induced by a chemotherapeutic agent. Insome embodiments, the chemotherapeutic agent is a vinca alkaloid. Insome embodiments, the vinca alkaloid is vincristine.

A wide variety of pharmaceuticals are known to cause drug-inducedneuropathy, including but not limited to anti-microbials,anti-neoplastic agents, cardiovascular drugs, hypnotics andpsychotropics, anti-rheumatics, and anti-convulsants.

Illustrative anti-microbials known to cause neuropathy include but arenot limited to isoniazid, ethambutol, ethionamide, nitrofurantoin,metronidazole, ciprofloxacin, chloramphenicol, thiamphenicol, diamines,colistin, streptomycin, nalidixic acid, clioquinol, sulphonamides,amphotericin, and penicillin.

Illustrative anti-neoplastic agents known to cause neuropathy includebut are not limited to procarbazine, nitrofurazone, podophyllum,mustine, ethoglucid, cisplatin, suramin, paclitaxel, chlorambucil,altretamine, carboplatin, cytarabine, docetaxel, dacarbazine, etoposide,ifosfamide with mesna, fludarabine, tamoxifen, teniposide, andthioguanine. Vinca alkaloids, such as vincristine, are known to beparticularly neurotoxic.

Illustrative cardiovascular drugs known to cause neuropathy include butare not limited to propranolol, perhexiline, hydrallazine, amiodarone,disopyramide, and clofibrate.

Illustrative hypnotics and psychotropics known to cause neuropathyinclude but are not limited to phenelzine, thalidomide, methaqualone,glutethimide, amitriptyline, and imipramine.

Illustrative anti-rheumatics known to cause neuropathy include but arenot limited to gold, indomethacin, colchicine, chloroquine, and phenylbutazone.

Illustrative anti-convulsants known to cause neuropathy include but arenot limited to phenytoin.

Other drugs known to cause neuropathy include but are not limited tocalcium carbimide, sulfoxone, ergotamine, propylthiouracil, sulthiame,chlorpropamide, methysergide, phenytoin, disulfiram, carbutamide,tolbutamide, methimazole, dapsone, and anti-coagulants.

The present disclosure contemplates combination therapies comprising theadministration of TBMs (alone or in combination with one or morearomatic-cationic peptides such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂) with one or moreadditional therapeutic regimens. The present disclosure also providescombination therapies comprising the administration of peptideconjugates of the present technology with one or more additionaltherapeutic regimens. In some embodiments, the additional therapeuticregimens are directed to the treatment or prevention of neuropathy orhyperalgesia or symptoms associated with neuropathy or hyperalgesia. Insome embodiments, the additional therapeutic regimens are directed tothe treatment or prevention of diseases or conditions unrelated toneuropathy or hyperalgesia. In some embodiments, the additionaltherapeutic regimens include regimens directed to the treatment orprevention of neuropathy or hyperalgesia or symptoms associated withneuropathy or hyperalgesia, in addition to diseases, conditions, orsymptoms unrelated to neuropathy or hyperalgesia or symptoms associatedwith neuropathy or hyperalgesia. In some embodiments, the additionaltherapeutic regimens comprise administration of one or more drugs,including but not limited to anti-microbials, anti-neoplastic agents,cardiovascular drugs, hypnotics and psychotropics, anti-rheumatics, andanti-convulsants. In embodiments, the additional therapeutic regimenscomprise non-pharmaceutical therapies, including but not limited todietary and lifestyle management.

In one aspect, the present disclosure provides a method for inhibitingor suppressing pain in a subject in need thereof, comprisingadministering to the subject an effective amount of TBMs (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂). In anotheraspect, the present disclosure provides a method for inhibiting orsuppressing pain in a subject in need thereof, comprising administeringto the subject an effective amount of peptide conjugates of the presenttechnology.

In some embodiments, TBMs (or derivatives, analogues, orpharmaceutically acceptable salts thereof) or peptide conjugates of thepresent technology (e.g., those including D-Arg-2′6′-Dmt-Lys-Phe-NH₂)are useful in suppressing pain through the binding and inhibition ofmu-opioid receptors. In other embodiments, TBMs (or derivatives,analogues, or pharmaceutically acceptable salts thereof) in combinationwith one or more active agents (e.g., an aromatic-cationic peptide suchas 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) will show a synergistic effect in thisregard.

Determination of the Biological Effect of Therapeutic BiologicalMolecules (TBMs) or Peptide Conjugates of the Present Technology

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific composition of thepresent technology and whether its administration is indicated fortreatment. In various embodiments, in vitro assays can be performed withrepresentative animal models, to determine if a given TBM (orderivatives, analogues, or pharmaceutically acceptable salts thereof)alone or in combination with one or more active agents (e.g., anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂), or a peptideconjugate-based therapeutic exerts the desired effect in treating adisease or condition. Compounds for use in therapy can be tested insuitable animal model systems including, but not limited to rats, mice,chicken, cows, monkeys, rabbits, and the like, prior to testing in humansubjects. Similarly, for in vivo testing, any of the animal model systemknown in the art can be used prior to administration to human subjects.

IV. Synthesis of Compositions of the Present Technology

The compounds useful in the methods of the present disclosure (e.g.,TBMs, or derivatives, analogues, or pharmaceutically acceptable saltsthereof) may be synthesized by any method known in the art.

The aromatic-cationic peptides disclosed herein (such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂) may be synthesized by any method known inthe art. Exemplary, non-limiting methods for chemically synthesizing theprotein include those described by Stuart and Young in “Solid PhasePeptide Synthesis,” Second Edition, Pierce Chemical Company (1984), andin “Solid Phase Peptide Synthesis,” Methods Enzymol. 289, AcademicPress, Inc, New York (1997).

Recombinant peptides may be generated using conventional techniques inmolecular biology, protein biochemistry, cell biology, and microbiology,such as those described in Current Protocols in Molecular Biology, Vols.I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach, Vols. Iand II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984);Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcriptionand Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture,Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986);Perbal, A Practical Guide to Molecular Cloning; the series, Meth.Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors forMammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu,Eds., respectively.

Aromatic-cationic peptide precursors may be made by either chemical(e.g., using solution and solid phase chemical peptide synthesis) orrecombinant syntheses known in the art. Precursors of e.g., amidatedaromatic-cationic peptides of the present technology may be made in likemanner. In some embodiments, recombinant production is believedsignificantly more cost effective. In some embodiments, precursors areconverted to active peptides by amidation reactions that are also knownin the art. For example, enzymatic amidation is described in U.S. Pat.No. 4,708,934 and European Patent Publications 0 308 067 and 0 382 403.Recombinant production can be used for both the precursor and the enzymethat catalyzes the conversion of the precursor to the desired activeform of the aromatic-cationic peptide. Such recombinant production isdiscussed in Biotechnology, Vol. 11 (1993) pp. 64-70, which furtherdescribes a conversion of a precursor to an amidated product. Duringamidation, a keto-acid such as an alpha-keto acid, or salt or esterthereof, wherein the alpha-keto acid has the molecular structureRC(O)C(O)OH, and wherein R is selected from the group consisting ofaryl, a C₁-C₄ hydrocarbon moiety, a halogenated or hydroxylated C₁-C₄hydrocarbon moiety, and a C₁-C₄ carboxylic acid, may be used in place ofa catalase co-factor. Examples of these keto acids include, but are notlimited to, ethyl pyruvate, pyruvic acid and salts thereof, methylpyruvate, benzoyl formic acid and salts thereof, 2-ketobutyric acid andsalts thereof, 3-methyl-2-oxobutanoic acid and salts thereof, and 2-ketoglutaric acid and salts thereof.

In some embodiments, the production of the recombinant aromatic-cationicpeptide may proceed, for example, by producing glycine-extendedprecursor in E. coli as a soluble fusion protein withglutathione-S-transferase. An α-amidating enzyme catalyzes conversion ofprecursors to active aromatic-cationic peptide. That enzyme isrecombinantly produced, for example, in Chinese Hamster Ovary (CHO)cells as described in the Biotechnology article cited above. Otherprecursors to other amidated peptides may be produced in like manner.Peptides that do not require amidation or other additionalfunctionalities may also be produced in like manner. Other peptideactive agents are commercially available or may be produced bytechniques known in the art.

V. Preparation of the Peptide Conjugates of the Present Technology

In some embodiments, at least one TBM and at least one aromatic-cationicpeptide as described herein, associate to form a peptide conjugate ofthe present technology. The TBM and aromatic-cationic peptide canassociate by any method known to those in the art. Suitable types ofassociations include chemical bonds and physical bonds. Chemical bondsinclude, for example, covalent bonds and coordinate bonds. Physicalbonds include, for instance, hydrogen bonds, dipolar interactions, vander Waal forces, electrostatic interactions, hydrophobic interactionsand aromatic stacking.

For a chemical bond or physical bond, a functional group on the TBMtypically associates with a functional group on the aromatic-cationicpeptide. Alternatively, a functional group on the aromatic-cationicpeptide associates with a functional group on the TBM.

The functional groups on the TBM and aromatic-cationic peptide canassociate directly. For example, a functional group (e.g., a sulfhydrylgroup) on a TBM can associate with a functional group (e.g., sulfhydrylgroup) on an aromatic-cationic peptide to form a disulfide.

Alternatively, the functional groups can associate through across-linking agent (i.e., linker). Some examples of cross-linkingagents are described below. The cross-linker can be attached to eitherthe TBM or the aromatic-cationic peptide.

The linker may and may not affect the number of net charges of thearomatic-cationic peptide. Typically, the linker will not contribute tothe net charge of the aromatic-cationic peptide. Each amino group, ifany, present in the linker will contribute to the net positive charge ofthe aromatic-cationic peptide. Each carboxyl group, if any, present inthe linker will contribute to the net negative charge of thearomatic-cationic peptide.

The number of TBMs or aromatic-cationic peptides in the peptideconjugate is limited by the capacity of the peptide to accommodatemultiple TBMs or the capacity of the TBM to accommodate multiplepeptides. For example, steric hindrance may hinder the capacity of thepeptide to accommodate especially large molecules. Alternatively, sterichindrance may hinder the capacity of the molecule to accommodate arelatively large (e.g., seven, eight, nine or ten amino acids in length)aromatic-cationic peptide.

The number of TBMs or aromatic-cationic peptides in the peptideconjugate is also limited by the number of functional groups present onthe other. For example, the maximum number of TBMs associated with apeptide conjugate depends on the number of functional groups present onthe aromatic-cationic peptide. Alternatively, the maximum number ofaromatic-cationic peptides associated with a TBM depends on the numberof functional groups present on the TBM.

In one embodiment, the peptide conjugate comprises at least one TBM, andin some embodiments, at least two TBMs, associated with anaromatic-cationic peptide. A relatively large peptide (e.g., eight, tenamino acids in length) containing several (e.g., 3, 4, 5 or more)functional groups can be associated with several (e.g., 3, 4, 5 or more)TBMs.

In another embodiment, the peptide conjugate comprises at least onearomatic-cationic peptide, and, in some embodiments, at least twoaromatic-cationic peptides, associated with a TBM. For example, a TBMcontaining several functional groups (e.g., 3, 4, 5 or more) can beassociated with several (e.g., 3, 4, or 5 or more) peptides.

In yet another embodiment, the peptide conjugate comprises onearomatic-cationic peptide associated to one TBM.

In one embodiment, a peptide conjugate comprises at least one TBMchemically bonded (e.g., conjugated) to at least one aromatic-cationicpeptide. The molecule can be chemically bonded to an aromatic-cationicpeptide by any method known to those in the art. For example, afunctional group on the TBM may be directly attached to a functionalgroup on the aromatic-cationic peptide. Some examples of suitablefunctional groups include, for example, amino, carboxyl, sulfhydryl,maleimide, isocyanate, isothiocyanate and hydroxyl.

The TBM may also be chemically bonded to the aromatic-cationic peptideby means of cross-linking agents, such as dialdehydes, carbodiimides,dimaleimides, and the like. Cross-linking agents can, for example, beobtained from Pierce Biotechnology, Inc., Rockford, Ill. The PierceBiotechnology, Inc. web-site can provide assistance. Additionalcross-linking agents include the platinum cross-linking agents describedin U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 of KreatechBiotechnology, B.V., Amsterdam, The Netherlands.

The functional group on the TBM may be different from the functionalgroup on the peptide. For example, if a sulfhydryl group is present onthe TBM, the TBM can be cross-linked to the peptide, e.g., [Dmt¹]DALDA,through the 4-amino group of lysine by using the cross-linking reagentSMCC (i.e., succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)from Pierce Biotechnology. In another example, the 4-amino group oflysine of DALDA can be conjugated directly to an alpha-phosphate groupon a TBM by using the crosslinking reagent EDC (i.e.,(N-[3-dimethylaminopropyl-N′-ethylcarboiimide]) from PierceBiotechnology.

Alternatively, the functional group on the TBM and peptide can be thesame. Homobifunctional cross-linkers are typically used to cross-linkidentical functional groups. Examples of homobifunctional cross-linkersinclude EGS (i.e., ethylene glycol bis[succinimidylsuccinate]), DSS(i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HC1),DTSSP (i.e., 3,3′-dithiobis[sulfosuccinimidylpropionate])), DPDPB (i.e.,1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane), and BMH (i.e.,bis-maleimidohexane). Such homobifunctional cross-linkers are alsoavailable from Pierce Biotechnology, Inc.

To chemically bond the TBMs and the peptides, the TBMs, peptides, andcross-linker are typically mixed together. The order of addition of theTBMs, peptides, and cross-linker is not important. For example, thepeptide can be mixed with the cross-linker, followed by addition of theTBM. Alternatively, the TBM can be mixed with the cross-linker, followedby addition of the peptide. Optimally, the TBM and the peptides aremixed, followed by addition of the cross-linker.

The chemically bonded peptide conjugates deliver the TBM and/oraromatic-cationic peptide to a cell. In some instances, the TBMfunctions in the cell without being cleaved from the aromatic-cationicpeptide. For example, if the aromatic-cationic peptide does not blockthe catalytic site of the molecule, then cleavage of the molecule fromthe aromatic-cationic peptide is not necessary.

In other instances, it may be beneficial to cleave the TBM from thearomatic-cationic peptide. The web-site of Pierce Biotechnology, Inc.described above can also provide assistance to one skilled in the art inchoosing suitable cross-linkers which can be cleaved by, for example,enzymes in the cell. Thus the molecule can be separated from thearomatic-cationic peptide. Examples of cleavable linkers include SMPT(i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene),Sulfo-LC-SPDP (i.e., sulfosuccinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e.,succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate),Sulfo-LC-SPDP (i.e., sulfosuccinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e.,N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP(i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

In another embodiment, a peptide conjugate comprises at least one TBMphysically bonded with at least one aromatic-cationic peptide. Anymethod known to those in the art can be employed to physically bond themolecules with the aromatic-cationic peptides.

For example, the aromatic-cationic peptides and TBMs can be mixedtogether by any method known to those in the art. The order of mixing isnot important. For instance, TBMs can be physically mixed with modifiedor unmodified aromatic-cationic peptides by any method known to those inthe art. Alternatively, the modified or unmodified aromatic-cationicpeptides can be physically mixed with the molecules by any method knownto those in the art.

For example, the aromatic-cationic peptides and TBMs can be placed in acontainer and agitated, by for example, shaking the container, to mixthe aromatic-cationic peptides and TBMs.

The aromatic-cationic peptides can be modified by any method known tothose in the art. For instance, the aromatic-cationic peptide may bemodified by means of cross-linking agents or functional groups, asdescribed above. The linker may and may not affect the number of netcharges of the aromatic-cationic peptide. Typically, the linker will notcontribute to the net charge of the aromatic-cationic peptide. Eachamino group, if any, present in the linker contributes to the netpositive charge of the aromatic-cationic peptide. Each carboxyl group,if any, present in the linker contributes to the net negative charge ofthe aromatic-cationic peptide.

For example, [Dmt₁]DALDA can be modified, through the 4-amino group oflysine by using the cross-linking reagent SMCC (i.e., succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) from PierceBiotechnology. To form a peptide conjugate, the modifiedaromatic-cationic peptide is usually formed first and then mixed withthe TBM.

One advantage of the physically bonded peptide conjugates, is that theTBM functions in a cell without the need for removing anaromatic-cationic peptide, such as those peptide conjugates in which theTBM is chemically bonded to an aromatic-cationic peptide. Furthermore,if the aromatic-cationic peptide does not block the catalytic site ofthe molecule, then dissociation of the complex is also not necessary.

In some embodiments, at least one TBM and at least one aromatic-cationicpeptide as described above (e.g., 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof), are associated to form aconjugate. The TBM and aromatic-cationic peptide can associate by anymethod known to those in the art. The following examples of peptide-TBMlinkages are provided by way of illustration only, and are not intendedto be limiting. In general, TBMs can be linked to an aromatic-cationicpeptide of the present disclosure by any suitable technique, withappropriate consideration of the need for pharmokinetic stability andreduced overall toxicity to the subject. A TBM can be coupled to anaromatic-cationic peptide either directly or indirectly (e.g., via alinker group).

Suitable types of associations include chemical bonds and physicalbonds. Chemical bonds include, for example, covalent bonds andcoordinate bonds. Physical bonds include, for instance, hydrogen bonds,dipolar interactions, van der Waal forces, electrostatic interactions,hydrophobic interactions and aromatic stacking. In some embodiments,bonds between the compounds are rapidly degraded or dissolved; in someembodiments, bonds are cleaved by drug metabolizing or excretorychemistry and/or enzymes.

For a chemical bond or physical bond, a functional group on the TBMtypically associates with a functional group on the aromatic-cationicpeptide. For example, TBMs may contain carboxyl functional groups, orhydroxyl functional groups. The free amine group of an aromatic-cationicpeptide may be cross-linked directly to the carboxyl group of a TBMusing 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDCor EDAC) or dicyclohexylcarbodiimide (DCC). Cross-linking agents can,for example, be obtained from Pierce Biotechnology, Inc., Rockford, Ill.The Pierce Biotechnology, Inc. website can provide assistance.

In some embodiments, a direct reaction between an additional activeagent (e.g., a TBM) and an aromatic-cationic peptide (e.g.,2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof), is formed when each possesses a functional group capable ofreacting with the other. Additionally or alternatively, a suitablechemical linker group can be used. A linker group can function as aspacer to distance the peptide and the TBM in order to avoidinterference with, for example, binding capabilities. A linker group canalso serve to increase the chemical reactivity of a substituent, andthus increase the coupling efficiency.

In exemplary embodiments, suitable linkage chemistries includemaleimidyl linkers and alkyl halide linkers (which react with asulfhydryl on the antibody moiety) and succinimidyl linkers (which reactwith a primary amine on the antibody moiety). Several primary amine andsulfhydryl groups are present on immunoglobulins, and additional groupscan be designed into recombinant immunoglobulin molecules. It will beevident to those skilled in the art that a variety of bifunctional orpolyfunctional reagents, both homo- and hetero-functional (such as thosedescribed in the catalogue of the Pierce Chemical Co., Rockford, Ill.),can be employed as a linker group. Coupling can be affected, e.g.,through amino groups, carboxyl groups, sulfhydryl groups or oxidizedcarbohydrate residues (see, e.g., U.S. Pat. No. 4,671,958).

As an additional or alternative coupling method, a TBM can be coupled tothe aromatic-cationic peptides disclosed herein, e.g., through anoxidized carbohydrate group at a glycosylation site, for example, asdescribed in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yet anotheralternative method of coupling an aromatic-cationic peptide to anadditional active agent is by the use of a non-covalent binding pair,such as streptavidin/biotin, or avidin/biotin. In these embodiments, onemember of the pair is covalently coupled to the aromatic-cationicpeptide, and the other member of the binding pair is covalently coupledto the TBM.

In some embodiments, a TBM may be more potent when free from thearomatic-cationic peptide, and it may be desirable to use a linker groupwhich is cleavable during or upon internalization into a cell, or whichis gradually cleavable over time in the extracellular environment. Anumber of different cleavable linker groups have been described.Examples of the intracellular release of active agents from these linkergroups include, e.g., but are not limited to, cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of aphotolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis ofderivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045), byserum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958),and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789).

In some embodiments the aromatic-cationic peptide, such as2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, is chemically linked to at least one TBM.In some embodiments, the peptide is linked to the TBM using a labilebond such that hydrolysis in vivo releases the two pharmaceuticallyactive agents. A schematic diagram illustrating exemplary embodiments isshown in FIG. 1. In some embodiments, the linkage comprises an ester, acarbonate, a carbamate or other labile linkage.

In some embodiments, an aromatic-cationic peptide as disclosed herein iscoupled to more than one TBM. For example, in some embodiments,aromatic-cationic peptide is coupled to a mixture of at least two TBMs.That is, more than one type of TBM can be coupled to onearomatic-cationic peptide. For instance, a TBM can be conjugated to anaromatic-cationic peptide to increase the effectiveness of the therapy,as well as lowering the required dosage necessary to obtain the desiredtherapeutic effect. Regardless of the particular embodiment,formulations with more than one moiety can be prepared in a variety ofways. For example, more than one moiety can be coupled directly to anaromatic-cationic peptide, or linkers that provide multiple sites forattachment (e.g., dendrimers) can be used. Alternatively, a carrier withthe capacity to hold more than one TBM can be used.

In some embodiments, linkers that that are cleaved within a cell mayalso be used. For example, heterocyclic “self-immolating” linkermoieties can be used to link aromatic-cationic peptides of the presenttechnology to TBMs (see, for example U.S. Pat. Nos. 7,989,434 and8,039,273, herein incorporated by reference in its entirety).

In some embodiments, the linker moiety comprises a heterocyclic“self-immolating moiety” bound to the aromatic-cationic peptide (e.g.,D-Arg, 2′6′-Dmt-Lys-Phe-NH₂) and a TBM and incorporates an amide groupor beta-glucuronide group that, upon hydrolysis by an intracellularprotease or beta-glucuronidase, initiates a reaction that ultimatelycleaves the self-immolative moiety from the aromatic-cationic peptidesuch that the TBM is released from the peptide in an active form.

Exemplary self-immolating moieties include those of Formulas presentedin FIG. 2. In FIG. 2, the wavy lines indicate the covalent attachmentsites to the aromatic-cationic peptide and the TBM, wherein:

U is O, S or NR⁶;

Q is CR⁴ or N;

V¹, V² and V³ are independently CR⁴ or N provided that for Formula Q andR of FIG. 2 at least one of Q, V¹ and V² is N;

T is NH, NR⁶, O or S pending from said drug moiety;

R¹, R², R³ and R⁴ are independently selected from H, F, Cl, Br, I, OH,—N(R⁵)₂, N(R⁵)₃ ⁺, C₁-C₈ alkylhalide, carboxylate, sulfate, sulfamate,sulfonate, —SO₂R⁵, —S(═O)R⁵, —SR⁵, —SO₂N(R⁵)₂, —C(═O)R⁵, —CO₂R⁵,—C(═O)N(R⁵)₂, —CN, —N₃, —NO₂, C₁-C₈ alkoxy, C₁-C₈halosubstituted alkyl,polyethyleneoxy, phosphonate, phosphate, C₁-C₈ alkyl, C₁-C₈ substitutedalkyl, C₂-C₈ alkenyl, C₂-C₈ substituted alkenyl, C₂-C₈ alkynyl, C₂-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₁-C₂₀heterocycle, and C₁-C₂₀ substituted heterocycle; or when taken together,R² and R³ form a carbonyl (═O), or spiro carbocyclic ring of 3 to 7carbon atoms; and

R⁵ and R⁶ are independently selected from H, C₁-C₈ alkyl, C₁-C₈substituted alkyl, C₂-C₈ alkenyl, C₂-C₈ substituted alkenyl, C₂-C₈alkynyl, C₂-C₈ substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substitutedaryl, C₁-C₂₀ heterocycle, and C₁-C₂₀ substituted heterocycle;

where C₁-C₈ substituted alkyl, C₂-C₈ substituted alkenyl, C₂-C₈substituted alkynyl, C₆-C₂₀ substituted aryl, and C₂-C₂₀ substitutedheterocycle are independently substituted with one or more substituentsselected from F, C₁, Br, I, OH, —N(R⁵)₂, —N(R⁵)₃ ⁺, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, C₁-C₈ alkylsulfonate, C₁-C₈alkylamino, 4-dialkylaminopyridinium, C₁-C₈ alkylhydroxyl, C₁-C₈alkylthiol, —SO₂R⁵, S(═O)R⁵, —SO₂N(R⁵)₂, —C(═O)R⁵, —CO₂R⁵, —C(═O)N(R⁵)₂,—CN, —N₃, NO₂, C₁-C₈ alkoxy, C₁-C₈ trifluoroalkyl, C₁-C₈ alkyl, C₃-C₁₂carbocycle, C₆-C₂₀ aryl, C₂-C₂₀ heterocycle, polyethyleneoxy,phosphonate, and phosphate.

The linker moiety may further include a cleavable peptide sequenceadjacent to the self-immolative moiety that is a substrate for anintracellular enzyme, for example a cathepsin such as cathepsin B, thatcleaves the cleavable peptide at the amide bond shared with theself-immolative moiety (e.g., Phe-Lys, Ala-Phe, or Val-Cit). In someembodiments, the amino acid residue chain length of the cleavablepeptide sequence ranges from that of a single amino acid to about eightamino acid residues. The following are exemplary enzymatically-cleavablepeptide sequences: Gly-Gly, Phe-Lys, Val-Lys, Phe-Phe-Lys,D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit,Trp-Cit, Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe, Gly-Val-Cit,Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N 9-tosyl-Arg, and Phe-N9-Nitro-Arg, in either orientation. Numerous specific cleavable peptidesequences suitable for use in the present formulations can be designedand optimized in their selectivity for enzymatic cleavage by aparticular intracellular enzyme, e.g., liver cell enzymes.

A spacer unit may be linked to the aromatic-cationic peptide via anamide, amine or thioether bond. In some embodiments, the TBM may beconnected to the self-immolative moiety of the linker via a chemicallyreactive functional group pending from the TBM. Exemplary schematics ofillustrative embodiments of such formulations are shown in FIG. 3.

In some embodiments, once the aromatic-cationic peptide-TBM conjugateenters the cell or blood stream, the linker is cleaved releasing thepeptide from the TBM. The formulations are not intended to be limited bylinkers or cleavage means. For example, in some embodiments, linkers arecleaved in the body (e.g., in the blood stream, interstitial tissue,gastrointestinal tract, etc.), releasing the peptide from the TBM viaenzymes (e.g., esterases) or other chemical reactions.

As explained above, an aromatic-cationic peptide can be linked to TBMsin a variety of ways, including covalent bonding either directly or viaa linker group, and non-covalent associations. For example, in someembodiments, the aromatic-cationic peptide and TBMs can be combined withencapsulation carriers. In some embodiments, this is especially usefulto allow the therapeutic compositions to gradually release thearomatic-cationic peptide and TBM over time while concentrating it inthe vicinity of the target cells.

In some embodiments, an aromatic-cationic peptide of the presenttechnology, e.g., 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, can be linked toa TBM of the present technology using an ester linkage. In someembodiments, the ester linkage is formed by coupling the pendanthydroxyl group of a TBM to a linker group bearing the formula:D-Arg-2′6′-Dmt-Lys-Phe-NH—(C═O)-(linker)-COOH

where linker may contain two or more carbon atoms.

As noted above, in some embodiments, the aromatic-cationic peptide-TBMconjugate is generated using a cleavable linker to facilitate release ofthe peptide in vivo. In some embodiments, the cleavable linker is anacid-labile linker, peptidase-sensitive linker, photolabile linker, adimethyl linker, or a disulfide-containing linker. In some embodiments,the linker is a labile linkage that is hydrolyzed in vivo to release theTBM and peptide. In some embodiments, the labile linkage comprises anester linkage, a carbonate linkage, or a carbamate linkage.

In some embodiments, the peptide aromatic-cationic peptide of thepresent technology, e.g., 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, is chemicallylinked to a TBM of the present technology using a labile linkage to forma pro-drug that upon hydrolysis in vivo releases the peptide and the TBMas active agents. In some embodiments, the labile linkage comprises anester linkage, a carbonate linkage, or a carbamate linkage.

As noted above, in one aspect, the present disclosure providescombination therapies for the treatment of disease or disorderscomprising administering an effective amount of aromatic-cationicpeptide-TBM conjugates that are linked via chemically labile bonds. Insome embodiments, the aromatic-cationic peptide-TBM conjugates will becreated by linking the aromatic-cationic peptide and the TBM via alinker group bearing the formula:HOOC-(linker)-COOH; orHOOC-(linker)-OH; orHOOC-(linker)-SH

where linker consists of one or more carbon atoms. In other embodiments,the linker consists of two or more carbon atoms.

By way of example, but not by way of limitation, FIG. 4 illustrates howstandard peptide chemistry can be used to form amide bonds between anaromatic-cationic peptide, such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, and the linkergroups described herein. Coupling between the aromatic-cationic peptideand the linker can be performed by any of the methods well-known in theart, including the use of carbodiimide coupling chemistry.

By way of example, but not by way of limitation, FIGS. 5A and 5Billustrate how standard esterification chemistry can be used to couple aTBM and a linker group using a labile ester linkage. Coupling betweenthe TBM and the linker can be performed by any of the methods well knownin the art, including the use of carbodiimide coupling chemistry.

Encapsulated Therapeutic Biological Molecules (TBMs) Linked toAromatic-Cationic Peptides

In some embodiments, at least one TBM is encapsulated before beinglinked to at least one aromatic-cationic peptide. By way of example, butnot by limitation, in some embodiments, at least one TBM is encapsulatedby a liposome or by polysaccharides, e.g., pectin or chitosan.

In some embodiments, at least one TBM is encapsulated by a liposome andthe aromatic-cationic peptide is linked to the outer surface of theliposome. In some embodiments, the liposome is modified to prolongcirculation, i.e., coated with polyethylene glycol (PEG). In someembodiments, the liposome is modified to improve targeting of theliposome, e.g., antibody conjugated liposomes.

Encapsulation of a TBM by liposomes can be performed by any methodsknown in the art. (See Nii, T. and Ishii, F., International Journal ofPharmaceutics, 298(11): 198-205 (2005)).

In some embodiments, at least one TBM is encapsulated by apolysaccharide and the aromatic-cationic peptide is linked to the outersurface of the polysaccharide. Examples of encapsulating polysaccharidesinclude, but are not limited to, pectin and chitosan.

Encapsulation of the TBM by polysaccharides can be performed by anymethods known in the art. (See Gan, Q. and Wang, T., Colloids andSurfaces B: Biointerfaces, 59(1): 24-34 (2007)).

In some embodiments, the TBM is encapsulated but not linked to thearomatic-cationic peptide.

VI. Modes of Administration

Any method known to those in the art for contacting a cell, organ ortissue with compositions such as peptide conjugates, TBMs, and/or anaromatic-cationic peptide such as 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, orpharmaceutically acceptable salt thereof, may be employed. Suitablemethods include in vitro, ex vivo, or in vivo methods.

In vitro methods typically include cultured samples. For example, a cellcan be placed in a reservoir (e.g., tissue culture plate), and incubatedwith a compound under appropriate conditions suitable for obtaining thedesired result. Suitable incubation conditions can be readily determinedby those skilled in the art.

Ex vivo methods typically include cells, organs or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the compound under appropriate conditions.The contacted cells, organs or tissues are typically returned to thedonor, placed in a recipient, or stored for future use. Thus, thecompound is generally in a pharmaceutically acceptable carrier.

In vivo methods typically include the administration of a TBM,aromatic-cationic peptide or peptide conjugate such as those describedherein, to a mammal such as a human. When used in vivo for therapy, anaromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology are administered to a mammal in an amount effective inobtaining the desired result or treating the mammal. The effectiveamount is determined during pre-clinical trials and clinical trials bymethods familiar to physicians and clinicians. The dose and dosageregimen will depend upon the degree of the infection in the subject, thecharacteristics of the particular aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology used, e.g., its therapeuticindex, the subject, and the subject's history.

An effective amount of an aromatic-cationic peptide, TBM, or peptideconjugate of the present technology useful in the present methods, suchas in a pharmaceutical composition or medicament, may be administered toa mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compositions or medicaments. Thearomatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may be administered systemically or locally.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regimen). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when an aromatic-cationic peptide, TBM, or peptide conjugate of thepresent technology contains both a basic moiety, such as an amine,pyridine or imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline, N,N′dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like. Salts derived from pharmaceuticallyacceptable inorganic acids include salts of boric, carbonic, hydrohalic(hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric,phosphoric, sulfamic, and sulfuric acids. Salts derived frompharmaceutically acceptable organic acids include salts of aliphatichydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic,malic, and tartaric acids), aliphatic monocarboxylic acids (e.g.,acetic, butyric, formic, propionic, and trifluoroacetic acids), aminoacids (e.g., aspartic and glutamic acids), aromatic carboxylic acids(e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic, hippuric, andtriphenylacetic acids), aromatic hydroxyl acids (e.g., o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(e.g., fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(e.g., benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, acetate, tartrate,trifluoroacetate, and the like.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology described herein can be incorporated into pharmaceuticalcompositions for administration, singly or in combination, to a subjectfor the treatment or prevention of a disorder described herein. Suchcompositions typically include the active agent and a pharmaceuticallyacceptable carrier. As used herein the term “pharmaceutically acceptablecarrier” includes saline, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Supplementary active compounds can also be incorporated into thecompositions.

Pharmaceutical compositions are typically formulated to be compatiblewith the intended route of administration. Routes of administrationinclude, for example, parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, respiratory (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfate; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates, and agents for the adjustment of tonicity, suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide. The preparationcan be enclosed in ampoules, disposable syringes or multiple-dose vialsmade of glass or plastic. For convenience of the patient or treatingphysician, the dosing formulation can be provided in a kit containingall necessary equipment (e.g., vials of drug, vials of diluent, syringesand needles) for a course of treatment (e.g., 7 days of treatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL® (BASF, Parsippany, N.J., USA) or phosphate buffered saline (PBS). Inall cases, a composition for parenteral administration must be sterileand should be formulated for ease of syringeability. The compositionshould be stable under the conditions of manufacture and storage, andmust be shielded from contamination by microorganisms such as bacteriaand fungi.

In one embodiment, the aromatic-cationic peptide, TBM, or peptideconjugate of the present technology are administered intravenously. Forexample, an aromatic-cationic peptide, TBM, or peptide conjugate of thepresent technology may be administered via rapid intravenous bolusinjection. In some embodiments, the aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology is administered as aconstant-rate intravenous infusion.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may also be administered orally, topically, intranasally,intramuscularly, subcutaneously, or transdermally. In one embodiment,transdermal administration is by iontophoresis, in which the chargedcomposition is delivered across the skin by an electric current.

Other routes of administration include intracerebroventricularly orintrathecally. Intracerebroventricularly refers to administration intothe ventricular system of the brain. Intrathecally refers toadministration into the space under the arachnoid membrane of the spinalcord. Thus, in some embodiments, intracerebroventricular or intrathecaladministration is used for those diseases and conditions which affectthe organs or tissues of the central nervous system.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may also be administered to mammals by sustained release, asis known in the art. Sustained release administration is a method ofdrug delivery to achieve a certain level of the drug over a particularperiod of time. The level is typically measured by serum or plasmaconcentration. A description of methods for delivering a compound bycontrolled release can be found in international PCT Application No. WO02/083106, which is incorporated herein by reference in its entirety.

Any formulation known in the art of pharmacy is suitable foradministration of the aromatic-cationic peptide, TBM, or peptideconjugate of the present technology. For oral administration, liquid orsolid formulations may be used. Examples of formulations includetablets, gelatin capsules, pills, troches, elixirs, suspensions, syrups,wafers, chewing gum and the like. The aromatic-cationic peptides, TBMs,or peptide conjugates of the present technology can be mixed with asuitable pharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

For systemic, intracerebroventricular, intrathecal, topical, intranasal,subcutaneous, or transdermal administration, formulations of thearomatic-cationic peptides, TBMs, or peptide conjugates of the presenttechnology may utilize conventional diluents, carriers, or excipientsetc., such as those known in the art to deliver the aromatic-cationicpeptides, TBMs, or peptide conjugates of the present technology. Forexample, the formulations may comprise one or more of the following: astabilizer, a surfactant, such as a nonionic surfactant, and optionallya salt and/or a buffering agent. The aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology may be delivered in the formof an aqueous solution, or in a lyophilized form.

The stabilizer may comprise, for example, an amino acid, such as forinstance, glycine; an oligosaccharide, such as, sucrose, tetralose,lactose; or a dextran. Alternatively, the stabilizer may comprise asugar alcohol, such as, mannitol. In some embodiments, the stabilizer orcombination of stabilizers constitutes from about 0.1% to about 10%weight for weight of the formulated composition.

In some embodiments, the surfactant is a nonionic surfactant, such as apolysorbate. Examples of suitable surfactants include Tween 20, Tween80; a polyethylene glycol or a polyoxyethylene polyoxypropylene glycol,such as Pluronic F-68 at from about 0.001% (w/v) to about 10% (w/v).

The salt or buffering agent may be any salt or buffering agent, such asfor example, sodium chloride, or sodium/potassium phosphate,respectively. In some embodiments, the buffering agent maintains the pHof the pharmaceutical composition in the range of about 5.5 to about7.5. The salt and/or buffering agent is also useful to maintain theosmolality at a level suitable for administration to a human or ananimal. In some embodiments, the salt or buffering agent is present at aroughly isotonic concentration of about 150 mM to about 300 mM.

Formulations of aromatic-cationic peptides, TBMs, or peptide conjugatesof the present technology may additionally contain one or moreconventional additives. Examples of such additives include a solubilizersuch as, for example, glycerol; an antioxidant such as for example,benzalkonium chloride (a mixture of quaternary ammonium compounds, knownas “quats”), benzyl alcohol, chloretone or chlorobutanol; an anestheticagent such as for example a morphine derivative; and an isotonic agentetc., such as described herein. As a further precaution againstoxidation or other spoilage, the pharmaceutical compositions may bestored under nitrogen gas in vials sealed with impermeable stoppers.

The mammal treated in accordance with the present technology may be anymammal, including, for example, farm animals, such as sheep, pigs, cows,and horses; pet animals, such as dogs and cats; and laboratory animals,such as rats, mice and rabbits. In one embodiment, the mammal is ahuman.

In some embodiments, aromatic-cationic peptides, TBMs, or peptideconjugates of the present technology are administered to a mammal in anamount effective in reducing the number of mitochondria undergoing, orpreventing, MPT. The effective amount is determined during pre-clinicaltrials and clinical trials by methods familiar to physicians andclinicians.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may be administered systemically or locally. In oneembodiment, the aromatic-cationic peptide, TBM, or peptide conjugate ofthe present technology are administered intravenously. For example,aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may be administered via rapid intravenous bolus injection. Inone embodiment, the aromatic-cationic peptide, TBM, or peptide conjugateof the present technology is administered as a constant-rate intravenousinfusion.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology can be injected directly into a coronary artery during, forexample, angioplasty or coronary bypass surgery, or applied ontocoronary stents.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology may include a carrier, which can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), or suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thiomerasol, and the like. Glutathione and other antioxidants can beincluded in the composition to prevent oxidation. In many cases, it isdesirable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialsmay be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology can be delivered in the formof an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of an aromatic-cationic peptide, TBM, or peptideconjugate of the present technology as described herein can also be bytransmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. In oneembodiment, transdermal administration may be performed byiontophoresis.

An aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology can be formulated in a carrier system. The carrier can be acolloidal system. The colloidal system can be a liposome, a phospholipidbilayer vehicle. In one embodiment, the therapeutic aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology isencapsulated in a liposome while maintaining protein integrity. As oneskilled in the art will appreciate, there are a variety of methods toprepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal.33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press(1993)). Liposomal formulations can delay clearance and increasecellular uptake (See Reddy, Ann. Pharmacother. 34 (78):915-923 (2000)).An active agent can also be loaded into a particle prepared frompharmaceutically acceptable ingredients including, but not limited to,soluble, insoluble, permeable, impermeable, biodegradable orgastroretentive polymers or liposomes. Such particles include, but arenot limited to, nanoparticles, biodegradable nanoparticles,microparticles, biodegradable microparticles, nanospheres, biodegradablenanospheres, microspheres, biodegradable microspheres, capsules,emulsions, liposomes, micelles and viral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.34:915-923 (2000)). A polymer formulation for human growth hormone (hGH)has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO96/40073 (Zale, et al.), and PCT publication WO 00/38651 (Shah, et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the aromatic-cationic peptides, TBMs, or peptideconjugates of the present technology are prepared with carriers thatwill protect the aromatic-cationic peptides, TBMs, or peptide conjugatesof the present technology against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylacetic acid. Suchformulations can be prepared using known techniques. The materials canalso be obtained commercially, e.g., from Alza Corporation (MountainView, Calif., USA) and Nova Pharmaceuticals, Inc. (Sydney, AU).Liposomal suspensions (including liposomes targeted to specific cellswith monoclonal antibodies to cell-specific antigens) can also be usedas pharmaceutically acceptable carriers. These can be prepared accordingto methods known to those skilled in the art, for example, as describedin U.S. Pat. No. 4,522,811.

The aromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology can also be formulated to enhance intracellular delivery. Forexample, liposomal delivery systems are known in the art. See, e.g.,Chonn and Cullis, Curr. Opin. Biotech. 6:698-708 (1995); Weiner,Immunometh. 4(3):201-9 (1994); Gregoriadis, Trends Biotechnol.13(12):527-37 (1995). Mizguchi, et al., Cancer Lett. 100:63-69 (1996),describes the use of fusogenic liposomes to deliver a protein to cellsboth in vivo and in vitro

Dosage, toxicity and therapeutic efficacy of the aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. In some embodiments, the aromatic-cationic peptides, TBMs, orpeptide conjugates of the present technology exhibit high therapeuticindices. While aromatic-cationic peptides, TBMs, or peptide conjugatesof the present technology that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For anyaromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology used in the methods described herein, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose can be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptide, TBM, orpeptide conjugate of the present technology, sufficient for achieving atherapeutic or prophylactic effect, range from about 0.000001 mg perkilogram body weight per day to about 10,000 mg per kilogram body weightper day. In some embodiments, the dosage ranges will be from about0.0001 mg per kilogram body weight per day to about 100 mg per kilogrambody weight per day. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight every day, every two days or every three days orwithin the range of 1-10 mg/kg every week, every two weeks or everythree weeks. In one embodiment, a single dosage of aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology ranges from0.1-10,000 micrograms per kg body weight. In one embodiment,aromatic-cationic peptide, TBM, or peptide conjugate concentrations in acarrier range from 0.2 to 2000 micrograms per delivered milliliter. Anexemplary treatment regimen entails administration once per day or oncea week. Intervals can also be irregular as indicated by measuring bloodlevels of glucose or insulin in the subject and adjusting dosage oradministration accordingly. In some methods, dosage is adjusted toachieve a desired fasting glucose or fasting insulin concentration. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, or until the subject shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regimen.

In some embodiments, a therapeutically effective amount ofaromatic-cationic peptide, TBM, or peptide conjugate of the presenttechnology is defined as a concentration of the aromatic-cationicpeptide, TBM, or peptide conjugate of the present technology at thetarget tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g., approximately 10⁻⁷ molar.This concentration may be delivered by systemic doses of 0.01 to 100mg/kg or equivalent dose by body surface area. The schedule of doses isoptimized to maintain the therapeutic concentration at the targettissue, such as by single daily or weekly administration, but alsoincluding continuous administration (e.g., parenteral infusion ortransdermal application).

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and thepresence of other diseases. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

Therapeutic Peptide Analogues

In some aspects, the present disclosure provides compositions includingTBMs or peptide conjugates of the present technology in combination withone or more active agents. In some embodiments, the active agentsinclude any one or more of the aromatic-cationic peptides shown inSection II. In some embodiments, the aromatic-cationic peptide is2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In some embodiments, the TBMs and aromatic-cationic peptides aremodified so as to increase resistance to enzymatic degradation. One wayof stabilizing peptides against enzymatic degradation is the replacementof an L-amino acid with a D-amino acid at the peptide bond undergoingcleavage. Peptide analogues are prepared containing one or more D-aminoacid residues. Another way to prevent enzymatic degradation isN-methylation of the α-amino group at one or more amino acid residues ofthe peptides. This will prevent peptide bond cleavage by any peptidase.Examples include: H-D-Arg-Dmt-Lys(N^(α)Me)-Phe-NH₂;H-D-Arg-Dmt-Lys-Phe(NMe)-NH₂; H-D-Arg-Dmt-Lys(N^(α)Me)-Phe(NMe)-NH₂; andH-D-Arg(N^(α)Me)-Dmt(NMe)-Lys(N^(α)Me)-Phe(NMe)-NH₂. N^(α)-methylatedanalogues have lower hydrogen bonding capacity and can be expected tohave improved intestinal permeability. In some embodiments, thetherapeutic peptide is modified by N-methylation of the α-amino group atone or more amino acid residues of the peptide.

An alternative way to stabilize a peptide amide bond (—CO—NH—) againstenzymatic degradation is its replacement with a reduced amide bond(Ψ[CH₂—NH]). This can be achieved with a reductive alkylation reactionbetween a Boc-amino acid-aldehyde and the amino group of the N-terminalamino acid residue of the growing peptide chain in solid-phase peptidesynthesis. The reduced peptide bond is predicted to result in improvedcellular permeability because of reduced hydrogen-bonding capacity.Examples include: H-D-Arg-Ψ[CH₂—NH]Dmt-Lys-Phe-NH₂,H-D-Arg-Dmt-Ψ[CH₂—NH]Lys-Phe-NH₂, H-D-Arg-Dmt-LysΨ[CH₂—NH]Phe-NH₂,H-D-Arg-Dmt-Ψ[CH₂—NH]Lys-Ψ[CH₂—NH]Phe-NH₂, etc. In some embodiments, thetherapeutic peptide is modified to include a reduced amide bond(Ψ[CH₂—NH]).

Stabilized peptide analogues may be screened for stability in plasma,simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Anamount of peptide is added to 10 ml of SGF with pepsin (COLE-PALMER®,Vernon Hills, Ill.) or SIF with pancreatin (COLE-PALMER®, Vernon Hills,Ill.), mixed and incubated for 0, 30, 60, 90 and 120 min. The samplesare analyzed by HPLC following solid-phase extraction. New analoguesthat are stable in both SGF and SIF are then be evaluated for theirdistribution across the Caco-2 monolayer. Analogues with apparentpermeability coefficient determined to be >10⁻⁶ cm/s (predictable ofgood intestinal absorption) will then have their activity in reducingmitochondrial oxidative stress determined in cell cultures.Mitochondrial ROS is quantified by FACS using MitoSox for superoxide,and HyPer-mito (a genetically encoded fluorescent indicator targeted tomitochondria for sensing H₂O₂). Mitochondrial oxidative stressors caninclude t-butylhydroperoxide, antimycin and angiotensin. Therapeuticpeptide analogues that satisfy all these criteria can then undergolarge-scale synthesis.

It is predicted that the proposed strategies will produce a therapeuticpeptide analog that would have oral bioavailability. The Caco-2 model isregarded as a good predictor of intestinal absorption by the drugindustry.

VII. Formulations

In some aspects, the present disclosure provide pharmaceuticalformulations for the delivery of aromatic-cationic peptides, TBMs, orpeptide conjugates of the present technology.

In one aspect, the present technology relates to a finishedpharmaceutical product adapted for oral delivery of TBM compositions orpeptide conjugates of the present technology, the product comprising:(a) a therapeutically effective amount of the active agent; (b) at leastone pharmaceutically acceptable pH-lowering agent; and (c) at least oneabsorption enhancer effective to promote bioavailability of the activeagent, wherein the pH-lowering agent is present in the finishedpharmaceutical product in a quantity which, if the product were added to10 milliliters of 0.1M aqueous sodium bicarbonate solution, would besufficient to lower the pH of the solution to no higher than 5.5, andwherein an outer surface of the product is substantially free of anacid-resistant protective vehicle.

In some embodiments, the pH-lowering agent is present in a quantitywhich, if the product were added to 10 milliliters of 0.1M sodiumbicarbonate solution, would be sufficient to lower the pH of thesolution to no higher than 3.5. In some embodiments, the absorptionenhancer is an absorbable or biodegradable surface active agent. In someembodiments, the surface active agent is selected from the groupconsisting of acylcarnitines, phospholipids, bile acids and sucroseesters. In some embodiments, the absorption enhancer is a surface activeagent selected from the group consisting of: (a) an anionic agent thatis a cholesterol derivative, (b) a mixture of a negative chargeneutralizer and an anionic surface active agent, (c) non-ionic surfaceactive agents, and (d) cationic surface active agents.

In some embodiments, the finished pharmaceutical product furthercomprises an amount of an additional peptide that is not aphysiologically active peptide effective to enhance bioavailability ofthe aromatic-cationic peptides, TBMs, or peptide conjugates of thepresent technology. In some embodiments, the finished pharmaceuticalproduct comprises at least one pH-lowering agent with a solubility inwater of at least 30 grams per 100 milliliters of water at roomtemperature. In some embodiments, the finished pharmaceutical productcomprises granules containing a pharmaceutical binder and, uniformlydispersed in the binder, the pH-lowering agent, the absorption enhancerand the aromatic-cationic peptides, TBMs, and/or peptide conjugates ofthe present technology.

In some embodiments, the finished pharmaceutical product comprises alamination having a first layer comprising at least one pharmaceuticallyacceptable pH-lowering agent and a second layer comprising thetherapeutically effective amount of the active agent (e.g., TBMs with orwithout aromatic-cationic peptides, or peptide conjugates); the productfurther comprising the at least one absorption enhancer effective topromote bioavailability of the active agent, wherein the first andsecond layers are united with each other, but the at least onepH-lowering agent and the active agent are substantially separatedwithin the lamination such that less than about 0.1% of the active agentcontacts the pH-lowering agent to prevent substantial mixing between thefirst layer material and the second layer material and thus to avoidinteraction in the lamination between the pH-lowering agent and theactive agent.

In some embodiments, the finished pharmaceutical product comprises apH-lowering agent selected from the group consisting of citric acid,tartaric acid and an acid salt of an amino acid. In some embodiments,the pH-lowering agent is selected from the group consisting ofdicarboxylic acids and tricarboxylic acids. In some embodiments, thepH-lowering agent is present in an amount not less than 300 milligrams.

VIII. Combination Therapy with Therapeutic Biological Molecule (TBM)Compositions and other Therapeutic Agents

In some embodiments, TBMs, aromatic-cationic peptides, peptideconjugates of the present technology or a combination thereof, may becombined with one or more additional therapeutic agents for theprevention, amelioration or treatment of a medical disease or condition.

In one embodiment, an additional therapeutic agent is administered to asubject in combination with a TBM, aromatic-cationic peptide, peptideconjugate of the present technology or a combination thereof, such thata synergistic therapeutic effect is produced.

The multiple therapeutic agents (including, but not limited to TBMs,aromatic-cationic peptides, or peptide conjugates of the presenttechnology) may be administered in any order or even simultaneously. Ifsimultaneously, the multiple therapeutic agents may be provided in asingle, unified form, or in multiple forms (by way of example only,either as a single pill or as two separate pills). One of thetherapeutic agents may be given in multiple doses, or both may be givenas multiple doses. If not simultaneous, the timing between the multipledoses may vary from more than zero weeks to less than four weeks. Inaddition, the combination methods, compositions and formulations are notto be limited to the use of only two agents.

IX. Examples

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way. For each of theexamples below, any aromatic-cationic peptide described herein could beused. By way of example, but not by limitation, the aromatic-cationicpeptide used in the examples below could be 2′6′-Dmt-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, or D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or any one or moreof the peptides shown in Section II and TBM is any compound shown inSection I.

Example 1: Compositions of the Present Technology Suppress OxidizedLow-Density Lipoprotein (oxLDL)-Induced CD36 Expression and Foam CellFormation in Mouse Peritoneal Macrophages

Atherosclerosis is thought to develop as a result of lipid uptake byvascular-wall macrophages leading to the development of foam cells andthe elaboration of cytokines and chemokines resulting in smoothmuscle-cell proliferation. CD36 is a scavenger receptor that mediatesuptake of oxLDL into macrophages and subsequent foam-cell development.CD36 knockout mice showed reduced uptake of oxLDL and reducedatherosclerosis. CD36 expression is regulated at the transcriptionallevel by various cellular stimuli, including glucose and oxLDL.

Macrophages are harvested from mice peritoneal cavity cultured overnightin the absence or presence of oxLDL (50 μg/mL) for 48 hours. Incubationwith oxLDL is anticipated to significantly increase CD36 mRNA. Inclusionof peptide conjugates (e.g., 10 nM-1 aromatic-cationic peptides (e.g.,an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the peptideconjugate treatment group), TBMs (e.g., an equivalent molar dose of TBMbased on the concentration of TBM administered in the peptide conjugatetreatment group), or TBMs in combination with aromatic-cationic peptides(e.g., an equivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) to the culture medium is anticipated to abolish theup-regulation of CD36.

Expression of CD36 protein, as determined by western blot, is alsoanticipated to significantly increase after a 48 hour incubation with 25μg/mL of oxLDL (oxLDL) when compared to vehicle control (V). Othercontrols will include CD36 expression from mouse heart (H) andmacrophages obtained from CD36 knockout mice (KO). The amount of CD36protein will be normalized to β-actin. Incubation with peptideconjugates, aromatic-cationic peptides, and TBMs alone or in combinationwith aromatic-cationic peptides is anticipated to significantly reduceCD36 protein levels compared to macrophages exposed to vehicle control(V). Incubation with peptide conjugates, aromatic-cationic peptides, orTBMs alone or in combination with aromatic-cationic peptides isanticipated to also significantly inhibit the up-regulation of CD36protein levels in macrophages exposed to 25 μg/mL oxLDL for 48 hours(oxLDL/S). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

Incubation of macrophages with oxLDL for 48 hours is also anticipated toincrease foam cell formation. Foam cell will be visualized by oil red O,which stains lipid droplets red. Inclusion of peptide conjugates,aromatic-cationic peptides, or TBMs alone or in combination witharomatic-cationic peptides is anticipated to prevent oxLDL-induced foamcell formation. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

Incubation of macrophages with oxLDL is anticipated to increase thepercentage of apoptotic cells. Treatment with peptide conjugates,aromatic-cationic peptides, or TBMs alone or in combination witharomatic-cationic peptides is anticipated to significantly reduce thepercentage of apoptotic cells induced by oxLDL. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBM (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating or preventingatherosclerosis in mammalian subjects.

Example 2: Compositions of the Present Technology Protect from theEffects of Acute Cerebral Ischemia

Cerebral ischemia initiates a cascade of cellular and molecular eventsthat lead to brain damage. One such event is post-ischemic inflammation.Using a mouse model of cerebral ischemia-reperfusion (20 minuteocclusion of the middle cerebral artery), it has been found that CD36 isup-regulated in microglia and macrophages in the post-ischemic brain,with increased reactive oxygen species production. CD36 knockout micehave a profound reduction in reactive oxygen species after ischemia andimproved neurological function compared to wild type mice.

Cerebral ischemia will be induced by occlusion of the right middlecerebral artery for 30 min. Wild-type (WT) mice will be given eithersaline vehicle (Veh) (i.p., n=9), peptide conjugates (2 mg/kg or 5mg/kg, i.p., n=6), aromatic-cationic peptides (e.g., an equivalent molardose of aromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (e.g., an equivalent molar dose of TBM based onthe concentration of TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) at 0, 6, 24 and 48 hours after ischemia. Mice will besacrificed 3 days after ischemia. Brains will be frozen, sectioned, andstained using Nissl stain. Infarct volume and hemispheric swelling willbe determined using an image analyzer. Data will be analyzed by one-wayANOVA with posthoc analysis.

It is anticipated that treatment of wild type mice with peptideconjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) at 0, 6, 24 and 48 hours after a 30 minuteocclusion of the middle cerebral artery will result in a significantreduction in infarct volume and hemispheric swelling compared to salinecontrols. It has previously been shown that thirty minutes of cerebralischemia in WT mice results in significant depletion in reducedglutathione (GSH) in the ipsilateral cortex and striatum compared to thecontralateral side in vehicle-treated animals. The depletion of GSH inthe ipsilateral cortex is anticipated to significantly be reduced whenthe mice are treated with peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) (2 mg/kgi.p. at 0, 6, 24 and 48 hours).

It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects with respect toprotecting subjects from the effects of acute cerebral ischemia comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBM in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating or preventingthe effects of acute cerebral ischemia in mammalian subjects.

Example 3: Compositions of the Present Technology Protect AgainstCD36-Mediated Acute Cerebral Ischemia

CD36 knockout (CD36 KO) mice will be subjected to acute cerebralischemia as described in Example 2. CD36 KO mice will be given eithersaline vehicle (Veh) (i.p., n=5), peptide conjugates (2 mg/kg or 5mg/kg, i.p., n=6), aromatic-cationic peptides (e.g., an equivalent molardose of aromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (e.g., an equivalent molar dose of TBM based onthe concentration of TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) at 0, 6, 24 and 48 hours following a 30 minute periodof ischemia. Infarct volume and hemispheric swelling in CD36 KO mice areexpected to be similar in subjects receiving saline, TBMs (alone or incombination with aromatic-cationic peptides), aromatic-cationic peptidesand peptide conjugates. It is expected that treatment of CD36 KO micewith peptide conjugates, aromatic-cationic peptides, or TBMs (with orwithout aromatic-cationic peptides) will fail to further prevent GSHdepletion in the ipsilateral cortex caused by the ischemia. The datawill show that the protective action of peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) in acute cerebral ischemia is a function of inhibition of CD36up-regulation.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing or treatingthe effects of CD36-mediated acute cerebral ischemia in mammaliansubjects.

Example 4: Compositions of the Present Technology Suppress CD36Expression in Post-Ischemic Brain

Transient occlusion of the middle cerebral artery has been shown tosignificantly increase the expression of CD36 mRNA in microglia andmacrophages in the post-ischemic brain. Wild-type mice will be givensaline vehicle (Veh, i.p., n=6), peptide conjugates (5 mg/kg, i.p.,n=6), aromatic-cationic peptides (e.g., an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (e.g., an equivalent molar dose of TBM based onthe concentration of TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) at 0 and 6 hours after a 30 minute period of ischemia.Levels of CD36 mRNA in post-ischemic brain will be determined using realtime PCR. It is anticipated that CD36 expression will be up-regulated asmuch as 6-fold in the ipsilateral brain compared to the contralateralbrain of mice receiving saline, with CD36 mRNA significantly reduced inthe ipsilateral brain of mice receiving peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for suppressing CD36expression in post-ischemic brain in mammalian subjects.

Example 5: Compositions of the Present Technology Suppress CD36Up-Regulation in Renal Tubular Cells Following Unilateral UreteralObstruction

Unilateral ureteral obstruction (UUO) is a common clinical disorderassociated with tubular cell apoptosis, macrophage infiltration, andinterstitial fibrosis. Interstitial fibrosis leads to a hypoxicenvironment and contributes to progressive decline in renal functiondespite surgical correction. CD36 has been shown to be expressed inrenal tubular cells.

UUO will be induced in Sprague-Dawley rats. The rats will be treatedwith saline (i.p., n=6), peptide conjugates (1 mg/kg i.p., n=6),aromatic-cationic peptides (e.g., an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (e.g., an equivalent molar dose of TBM based onthe concentration of TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) one day prior to induction of UUO, and once daily for14 days after UUO induction. Rats will be sacrificed and the kidneysremoved, embedded in paraffin, and sectioned. The sections will betreated with an anti-CD36 polyclonal IgG (Santa Cruz, sc-9154; diluted1:100 with blocking serum) at room temperature for 1.5 hours. The slideswill then be incubated with the second antibody conjugated with biotin(anti-rabbit IgG-B1; ABC kit, PK-6101) at room temperature for 30 min.The slides will then be treated with avidin, developed with DAB andcounterstained with 10% hematoxylin. The contralateral unobstructedkidney will serve as the control for each animal.

It is anticipated that UUO will result in tubular dilation andsignificant increase in expression of CD36 in the tubular cells ofsaline-treated subjects. Tubular dilation is also anticipated in ratstreated with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides). But it is anticipated thattreatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will result in asignificant reduction in CD36 expression. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

To demonstrate that peptide conjugates reduce lipid peroxidation inkidney after UUO, rats will be treated with either saline (n=6), peptideconjugates (1 mg/kg i.p., n=6), aromatic-cationic peptides (e.g., anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group), TBMs (e.g., an equivalent molar doseof TBM based on the concentration of TBM administered in the peptideconjugate treatment group), or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) one day prior toinduction of UUO, and once daily for 14 days after UUO. Rats will thenbe sacrificed, kidneys removed, embedded in paraffin and sectioned.Slides will be incubated with anti-HNE rabbit IgG and a biotin-linkedanti-rabbit IgG will be used as secondary antibody. The slides will bedeveloped with DAB. Lipid peroxidation, which is increased by UUO, isanticipated to be reduced by treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

It is anticipated that HNE stain (brown) will be significantly increasedin tubular cells in the obstructed kidney compared to the contralateralcontrol. It is anticipated that obstructed kidneys from rats treatedwith peptide conjugates, aromatic-cationic peptides, or TBMs (with orwithout aromatic-cationic peptides) will show significantly less HNEstaining compared to saline-treated rats. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

To demonstrate that peptide conjugates reduce tubular cell apoptosis inobstructed kidney after UUO, rats will be treated with either saline(n=6), peptide conjugates (1 mg/kg i.p., n=6), aromatic-cationicpeptides (e.g., an equivalent molar dose of aromatic-cationic peptidebased on the concentration of the aromatic-cationic peptide administeredin the peptide conjugate treatment group), TBMs (e.g., an equivalentmolar dose of TBM based on the concentration of TBM administered in thepeptide conjugate treatment group), or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) one day prior toinduction of UUO, and once daily for 14 days after UUO. Rats will thenbe sacrificed, kidneys removed, embedded in paraffin and sectioned. Toquantify nuclei with fragmented DNA, TUNEL assay will be performed within situ TUNEL kit. Slides will be developed with DAB and counterstainedwith 10% hematoxylin. The up-regulation of CD36 in saline-treatedcontrols associated with tubular cell apoptosis is anticipated to besignificantly inhibited by treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that there will be a significant increasein apoptotic cells observed in the obstructed kidney from saline-treatedanimals when compared to the contralateral unobstructed control. Thenumber of apoptotic cells is anticipated to be significantly reduced inobstructed kidney from animals treated with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

Macrophage infiltration and interstitial fibrosis are anticipated to beprevented by treatment with peptide conjugates, aromatic-cationicpeptides, or TBMs (alone or in combination with aromatic-cationicpeptides). Rats will be treated with either saline (n=6), peptideconjugates (1 mg/kg i.p., n=6), aromatic-cationic peptides (e.g., anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group), TBMs (e.g., an equivalent molar doseof TBM based on the concentration of TBM administered in the peptideconjugate treatment group) or TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate treatmentgroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate treatment group) one day prior to induction of UUO,and once daily for 14 days after UUO. Rats will then be sacrificed, thekidneys removed, embedded in paraffin and sectioned. Slides will betreated with monoclonal antibody for ED1 macrophage (1:75; Serotec).Horseradish peroxidase-linked rabbit anti-mouse secondary antibody(Dako) will be used for macrophage detection. Sections will then becounterstained with 10% hematoxylin. The number of macrophages in theobstructed kidney in saline-treated rats is anticipated to besignificantly increased compared to the contralateral unobstructedcontrol. Macrophage infiltration is anticipated to be significantlyreduced in rats treated with peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides). It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

Rats will be treated with either saline (n=6), peptide conjugates (1mg/kg i.p., n=6), aromatic-cationic peptides (e.g., an equivalent molardose of aromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (e.g., an equivalent molar dose of TBM based onthe concentration of TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) one day prior to induction of UUO, and once daily for14 days after UUO. Rats will then be sacrificed, kidneys removed,embedded in paraffin and sectioned. Slides will be stained withhematoxylin and eosin and Masson's trichrome for interstitial fibrosis(blue stain). It is anticipated that obstructed kidneys fromsaline-treated rats will show increased fibrosis compared to thecontralateral unobstructed control, while obstructed kidneys from ratstreated with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will show significantlyless fibrosis. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) suppressthe up-regulation of CD36 in renal tubular cells induced by UUO. Theseresults will further show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for suppressing theup-regulation of CD36 in renal tubular cells induced by UUO in mammaliansubjects.

Example 6: Compositions of the Present Technology Suppress CD36Up-Regulation in Isolated Hearts Upon Reperfusion after Prolonged ColdIschemic Storage

Organ transplantation requires hypothermic storage of the isolated organfor transport to the recipient. Currently, cardiac transplantation islimited by the short time of cold ischemic storage that can be toleratedbefore coronary blood flow is severely compromised (<4 hours). Theexpression of CD36 in coronary endothelium and cardiac muscles isup-regulated in isolated hearts subjected to prolonged cold ischemicstorage and warm reperfusion.

Isolated guinea pig hearts will be perfused with St. Thomas solutionalone or St. Thomas solution containing peptide conjugates (1-100 nM),aromatic-cationic peptides (e.g., an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (e.g., an equivalent molar dose of TBM based onthe concentration of TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) for 3 minutes and then stored in the same solution at4° C. for 18 hours. After ischemic storage, hearts will be re-perfusedwith 34° C. Krebs-Henseleit solution for 90 min. Hearts freshly isolatedfrom guinea pigs will be used as controls.

The hearts will be fixed in paraffin and sliced for immunostaining withan anti-CD36 rabbit polyclonal antibody. It is anticipated that thesections from a representative heart stored in St. Thomas solution for18 hours at 4° C. will show increased CD36 staining compared to freshlyisolated controls. CD36 staining is anticipated to be significantlyreduced in hearts stored with peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) in St.Thomas solution for 18 hours. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is also anticipated that there will be a decrease in lipidperoxidation in the hearts treated with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). Guinea pig hearts will be perfused with a cardioplegicsolution (St. Thomas solution) alone or St. Thomas solution containing1-100 nM peptide conjugates, aromatic-cationic peptides (e.g., anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group), TBMs (e.g., an equivalent molar doseof TBM based on the concentration of TBM administered in the peptideconjugate treatment group), or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) for 3 minutes andthen subjected to 18 hours of cold ischemia (4° C.). The hearts will bethen re-perfused with Krebs Henseleit buffer at 34° C. for 90 minutes.Immunohistochemical analysis of 4-hydroxynonenol (HNE)-modified proteinsin paraffin sections from tissue slices will be performed by incubationwith an anti-HNE antibody (Santa Cruz) and a fluorescent secondaryantibody. HNE staining is anticipated to significantly increase inhearts subjected to 18 hours of cold storage in St. Thomas solutioncompared to non-ischemic hearts. HNE staining is anticipated to bereduced in hearts stored in peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) comparedto controls stored in St. Thomas solution alone. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

Further, it is anticipated that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) willdramatically reduce endothelial apoptosis. Guinea pig hearts will beperfused with St. Thomas solution alone or St. Thomas solutioncontaining peptide conjugates, aromatic-cationic peptides (e.g., anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group), TBMs (e.g., an equivalent molar doseof TBM based on the concentration of TBM administered in the peptideconjugate treatment group), or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) for 3 minutes andthen subjected to 18 hours of cold ischemia (4° C.). The hearts willthen be re-perfused with Krebs-Henseleit buffer at 34° C. for 90 min.After deparaffinization, sections will be incubated withdeoxynucleotidyl transferase (Tdt) with digoxigenin-dNTP for 1 hour. Thereaction will be stopped with terminating buffer. A fluorescentanti-digoxigenin antibody will then be applied.

It is anticipated that hearts subjected to 18 hours of cold storage inSt. Thomas solution will show prominent endothelial apoptosis, whereasno endothelial apoptosis will be observed in non-ischemic controlhearts. It is anticipated that apoptotic cells will not be observed inhearts stored in peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides). It is anticipated that asignificant improvement of coronary blood flow after prolonged coldischemic storage and warm reperfusion will occur when hearts arepreserved in peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides).

It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects with respect tosuppressing CD36 up-regulation in isolated organs upon reperfusionfollowing prolonged cold ischemic storage compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for suppressing CD36up-regulation in isolated organs upon reperfusion following prolongedcold ischemic storage.

Example 7: Compositions of the Present Technology Prevent Renal Damagein Diabetic Mice

CD36 expression is up-regulated in a variety of tissues of diabeticpatients, including monocytes, heart, kidneys, and blood. High glucoseis known to up-regulate the expression of CD36 by improving thetranslational efficiency of CD36 mRNA. Diabetic nephropathy is a commoncomplication of type 1 and type 2 diabetes, and is associated withtubular epithelial degeneration and interstitial fibrosis. CD36 has beenidentified as a mediator of tubular epithelial apoptosis in diabeticnephropathy. High glucose stimulates CD36 expression and apoptosis inproximal tubular epithelial cells.

Streptozotocin (STZ) will be used to induce diabetes in mice. Fivegroups of CD-1 mice will be studied: Group I—no STZ treatment; GroupII—STZ (50 mg/kg, i.p.) will be given once daily for 5 days; GroupIII—STZ (50 mg/kg, i.p.) will be given once daily for 5 days, + peptideconjugates (3 mg/kg, i.p.) will be given once daily for 16 days; GroupIV—STZ (50 mg/kg, i.p.) will be given once daily for 5 days, +aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group) will be given once daily for 16 days; Group V—STZ (50mg/kg, i.p.) will be given once daily for 5 days, + TBM (an equivalentmolar dose of TBM based on the concentration of the TBM administered inthe peptide conjugate treatment group) will be given once daily for 16days; Group VI—STZ (50 mg/kg, i.p.) will be given once daily for 5days, + TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will be given once daily for 16 days. It is anticipatedthat STZ treatment will result in a progressive increase in bloodglucose. Animals will be sacrificed after 3 weeks and kidney tissuespreserved for histopathology. Kidney sections will be examined byPeriodic Schiff (PAS) staining for renal tubular brush border.

It is anticipated that STZ treatment will cause a dramatic loss of brushborder in proximal tubules of the renal cortex, with tubular epithelialcells showing small condensed nuclei. It is anticipated that dailytreatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will prevent the loss ofbrush border in the STZ-treated mice, and the tubular epithelial nucleiwill appear normal. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

It is anticipated that STZ treatment will induce significant apoptosisin tubular epithelial cells. Kidney sections will be examined forapoptosis using a TUNEL assay as described herein. It is anticipatedthat kidney sections from mice treated with STZ will show a large numberof apoptotic nuclei in the proximal tubules, compared to non-STZ treatedcontrols. It is anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will dramatically reduce apoptotic cells in the proximaltubule CD36 expression in proximal tubular epithelial cells. It isanticipated that by reducing CD36 expression, peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will inhibit tubular cell apoptosis and the loss of brushborder in mice treated with STZ, without affecting blood glucose levels.It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBM in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating or preventingrenal damage in diabetic mammals.

Example 8: Penetration of Cell Membranes by Compositions of the PresentTechnology

The cellular uptake of [³H] TBMs (with or without aromatic-cationicpeptides) or [³H] peptide conjugates will be studied using Caco-2 cells(human intestinal epithelial cells), and confirmed using SH-SY5Y (humanneuroblastoma), HEK293 (human embryonic kidney) and CRFK (kidneyepithelial) cells. Monolayers of cells will be cultured in 12-wellplates (5×10⁵ cells/well) coated with collagen for 3 days. On day 4, thecells will be washed twice with pre-warmed HBSS, and incubated with 0.2mL of HBSS containing 250 nM [³H] peptide conjugates; [³H]aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); [³H] TBMs (an equivalent molar dose of TBM based onthe concentration of the TBM administered in the peptide conjugatetreatment group); or TBMs in combination with aromatic-cationic peptides(e.g., an equivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) at 37° C. or 4° C. for various times up to 1 hour.

It is anticipated that [³H] TBMs (with or without aromatic-cationicpeptides) or [³H] peptide conjugates will be observed in cell lysate andsteady state levels will be achieved within 1 hour. It is anticipatedthat the rate of [³H] TBMs (with or without aromatic-cationic peptides)or [³H] peptide conjugate uptake will be slower at 4° C. compared to 37°C., but that uptake will reach a high level of saturation by 45 minutes(e.g., 76.5%) and a higher level of saturation by 1 hour (e.g., 86.3%).It is anticipated that the internalization of [³H] TBMs (with or withoutaromatic-cationic peptides) or [³H] peptide conjugates will not belimited to Caco-2 cells, and that similar results will be achieved withSH-SY5Y, HEK293 and CRFK cells. The intracellular concentration of TBMs(with or without aromatic-cationic peptides) or peptide conjugates isanticipated to be approximately 50 times higher than the extracellularconcentration following 1 hour of incubation. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects with respect to cell membrane permeability comparedto treatment with aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

In a separate experiment, cells will be incubated with a range ofpeptide conjugate concentrations (1 μM-3 mM); aromatic-cationic peptides(an equivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group); or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) for 1 hour at 37°C. At the end of the incubation period, cells will be washed 4 timeswith HBSS, and 0.2 mL of 0.1N NaOH with 1% SDS will be added to eachwell. The cell lysates will then be transferred to scintillation vialsand radioactivity will be counted. To distinguish between internalizedradioactivity and surface-associated radioactivity, an acid-wash stepwill be included. Prior to cell lysis, cells will be incubated with 0.2mL of 0.2 M acetic acid/0.05 M NaCl for 5 minutes on ice.

The uptake of TBMs (with or without aromatic-cationic peptides) orpeptide conjugates into Caco-2 cells will be confirmed by confocal laserscanning microscopy (CLSM) using a fluorescent analog of TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates. Cells will begrown as described above and will be plated on (35 mm) glass dishes(MatTek Corp., Ashland, Mass.) for 2 days. The medium will then beremoved and cells will be incubated with 1 mL of HBSS containing 0.1 μMto 1.0 μM of the fluorescent analog of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates at 37° C. for 1 hour.Cells will be washed three times with ice-cold HBSS and covered with 200μL of PBS. Microscopy will be performed within 10 minutes at roomtemperature using a Nikon confocal laser scanning microscope with aC-Apochromat 63×/1.2 W corr objective. Excitation will be performed at340 nm by means of a UV laser, and emission will be measured at 520 nm.For optical sectioning in z-direction, 5-10 frames with 2.0μ z-stepswill be collected.

CLSM will be used to confirm the uptake of fluorescent TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates into Caco-2cells after incubation with 0.1 μM fluorescent analog for 1 h at 37° C.It is anticipated that the uptake of the fluorescent analog will besimilar at 37° C. and 4° C. It is anticipated that the fluorescence willappear diffuse throughout the cytoplasm but will be completely excludedfrom the nucleus.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for penetrating cellmembranes.

Example 9: Targeting of Compositions of the Present Technology toMitochondria In Vivo

A fluorescent analog of TBMs (with or without aromatic-cationicpeptides) or peptide conjugates will be prepared. The cells will begrown as described above and will be plated on (35 mm) glass dishes(MatTek Corp., Ashland, Mass.) for 2 days. The medium will be thenremoved and cells will be incubated with 1 mL of HBSS containing 0.1 μMfluorescent analog at 37° C. for 15 minutes to 1 hour.

Cells will also be incubated with tetramethylrhodamine methyl ester(TMRM, 25 nM), a dye for staining mitochondria, for 15 minutes at 37° C.Cells will be washed three times with ice-cold HBSS and covered with 200μL of PBS. Microscopy will be performed within 10 minutes at roomtemperature using a Nikon confocal laser scanning microscope with aC-Apochromat 63×/1.2 W corr objective.

For fluorescent analog, excitation will be performed at 350 nm using aUV laser, and emission will be measured at 520 nm. For TMRM, excitationwill be performed at 536 nm, and emission will be measured at 560 nm.

It is anticipated that CLSM will show the uptake of fluorescent analoginto Caco-2 cells after incubation for as little as 15 minutes at 37°C., and that staining will be excluded from the nucleus. Mitochondriallocalization of fluorescent analog will be demonstrated by the overlapof the fluorescent analog and TMRM.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods comprising the targetingof the compound to mitochondria in vivo.

Example 10: Targeting of Compositions of the Present Technology toIsolated Mitochondria

To isolate mitochondria from mouse liver, mice will be sacrificed bydecapitation. The liver will be removed and rapidly placed into chilledliver homogenization medium. The liver will be finely minced usingscissors and then homogenized by hand using a glass homogenizer.

The homogenate will be centrifuged for 10 minutes at 1000×g at 4° C. Thesupernatant will be aspirated and transferred to polycarbonate tubes andcentrifuged again for 10 minutes at 3000×g, 4° C. The resultingsupernatant will be removed, and the fatty lipids on the side-wall ofthe tube will be removed.

The pellet will be resuspended in liver homogenate medium and thehomogenization repeated twice. The final purified mitochondrial pelletwill be resuspended in medium. Protein concentration in themitochondrial preparation will be determined by the Bradford procedure.

Approximately 1.5 mg mitochondria in 400 μL buffer will be incubatedwith [³H] peptide conjugates; [³H] aromatic-cationic peptides (anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); [³H] TBMs (an equivalent molar doseof TBM based on the concentration of the TBM administered in the peptideconjugate treatment group); or [³H] TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) for 5-30 minutesat 37° C. The mitochondria will then be centrifuged and the amount ofradioactivity will be determined in the mitochondrial fraction andbuffer fraction. Assuming a mitochondrial matrix volume of 0.7 μL/mgprotein (Lim, et al., J. Physiol. 545:961-974 (2002)), it is anticipatedthat the concentration of [³H] TBMs (with or without aromatic-cationicpeptides) or [³H] peptide conjugates in mitochondria will be higher thanin the buffer, indicating that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates are concentrated in mitochondria.

To demonstrate that TBMs (with or without aromatic-cationic peptides) orpeptide conjugates are selectively distributed to mitochondria, we willexamine the uptake of fluorescent TBMs (with or withoutaromatic-cationic peptides) or fluorescent peptide conjugates and [³H]TBMs (with or without aromatic-cationic peptides) or [³H] peptideconjugates into isolated mouse liver mitochondria. The rapid uptake offluorescent TBMs (with or without aromatic-cationic peptides) orfluorescent peptide conjugates is anticipated. Pre-treatment ofmitochondria with carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone(FCCP), an uncoupler that results in immediate depolarization ofmitochondria, is anticipated to reduce the uptake of fluorescent TBMs(with or without aromatic-cationic peptides) or fluorescent peptideconjugates, demonstrating that the uptake is membranepotential-dependent.

To demonstrate that the mitochondrial targeting is not an artifact ofthe fluorophore, we will also examine mitochondrial uptake of [³H]peptide conjugates or [³H] TBMs (with or without aromatic-cationicpeptides). Isolated mitochondria will be incubated with [³H] peptideconjugates or [³H] TBMs (with or without aromatic-cationic peptides) andradioactivity will be determined in the mitochondrial pellet andsupernatant. It is anticipated that the amount of radioactivity in thepellet will not change from 2 minutes to 8 minutes, and that treatmentof mitochondria with FCCP will decrease the amount of [³H] peptideconjugates or [³H] TBMs (with or without aromatic-cationic peptides)associated with the mitochondrial pellet.

The minimal effect of FCCP on mitochondrial uptake of TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates will show that[³H] TBMs (with or without aromatic-cationic peptides) or [³H] peptideconjugates are likely associated with mitochondrial membranes or in theinter-membrane, space rather than in the mitochondrial matrix. We willalso demonstrate the effect of mitochondrial swelling on themitochondrial localization of fluorescent TBMs (with or withoutaromatic-cationic peptides) or fluorescent peptide conjugates usingalamethicin to induce swelling and rupture of the outer mitochondrialmembrane. It is anticipated that the uptake of fluorescent TBMs (with orwithout aromatic-cationic peptides) or fluorescent peptide conjugateswill be only partially reversed by mitochondrial swelling. This resultwill confirm that TBMs (with or without aromatic-cationic peptides) orpeptide conjugates are associated with mitochondrial membranes.

It is further anticipated that treatment with the peptide conjugate willshow a synergistic effect with respect to mitochondrial targetingcompared to treatment with aromatic-cationic peptides or TBMs (alone orin combination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods comprising the targetingof the TBMs (with or without aromatic-cationic peptides) or peptideconjugates to isolated mitochondria.

Example 11: Compositions of the Present Technology do not AlterMitochondrial Respiration or Membrane Potential

This Example will demonstrate that TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates do not altermitochondrial function, as measured by oxygen consumption andmitochondrial membrane potential.

Isolated mouse liver mitochondria will be incubated with 100 pM of TBMs(with or without aromatic-cationic peptides) or peptide conjugates, andoxygen consumption will be measured. It is anticipated that TBMs (withor without aromatic-cationic peptides) or peptide conjugates will notalter oxygen consumption during state 3 or state 4, or the respiratoryratio (state 3/state 4) (6.2 versus 6.0). Mitochondrial membranepotential will be measured using TMRM. It is anticipated that additionof mitochondria will result in immediate quenching of the TMRM signal,which will be readily reversible by the addition of FCCP, indicatingmitochondrial depolarization. It is anticipated that the addition ofCa²⁺ (150 μM) will result in immediate mitochondrial depolarizationfollowed by progressive loss of quenching indicative of MPT. It isanticipated that the addition of TBMs (with or without aromatic-cationicpeptides) or peptide conjugates alone, even at 200 μM, will not causemitochondrial depolarization or MPT.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, do not alter mitochondrial function, as measuredby oxygen consumption and mitochondrial membrane potential.

Example 12: Compositions of the Present Technology Protect Against MPTInduced by Ca²⁺ and 3NP

This Example will demonstrate that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) protectagainst MPT induced by Ca²⁺ overload and 3-nitropropionic acid (3NP).

It is anticipated that the pre-treatment of isolated mitochondria with10 μM peptide conjugates, aromatic-cationic peptides (an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofthe aromatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) for 2 minutes prior to addition of Ca²⁺ will resultonly in transient depolarization and will prevent the onset of MPT. Itis further anticipated that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) willdose-dependently increase the tolerance of mitochondria to cumulativeCa²⁺ challenges. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

3-Nitropropionic acid (3NP) is an irreversible inhibitor of succinatedehydrogenase in complex II of the electron transport chain. It isanticipated that the addition of 3NP (1 mM) to isolated mitochondriawill cause the loss of mitochondrial membrane potential and the onset ofMPT. It is further anticipated that the pre-treatment of mitochondriawith peptide conjugates, aromatic-cationic peptides, or TBMs (with orwithout aromatic-cationic peptides) will dose-dependently delay theonset of MPT induced by 3NP. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

Caco-2 cells will be treated with 3NP (10 mM) alone or in the presenceof peptide conjugates (0.1 μM); aromatic-cationic peptides (anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group); or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) for 4 hours, andthen incubated with TMRM and examined by CLSM. It is expected that3NP-treated cells will display reduced fluorescence compared to controlcells, which indicates mitochondrial depolarization. By contrast, it isanticipated that concurrent treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will protect against mitochondrial depolarization caused by3NP. It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBM in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for protectingmitochondria against MPT in vitro or in vivo.

Example 13: Compositions of the Present Technology Protect AgainstMitochondrial Swelling and Cytochrome c Release

MPT pore opening results in mitochondrial swelling. We will demonstratethe effects of peptide conjugates, aromatic-cationic peptides, or TBMsalone or in combination with aromatic-cationic peptides on mitochondrialswelling by measuring reduction in absorbance at 540 nm (A540).Mitochondrial suspensions will be centrifuged and the amount ofcytochrome c in the pellet and supernatant will be determined using acommercially available ELISA kit. It is anticipated that thepre-treatment of isolated mitochondria with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will inhibit swelling and cytochrome c release induced by Ca²⁺overload. It is further anticipated that in addition to preventing MPTinduced by Ca²⁺ overload, peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) will alsoprevent mitochondrial swelling induced by 1-methyl-4-phenylpyridium ions(MPP⁺), an inhibitor of complex I of the mitochondrial electrontransport chain. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for protectingmitochondria against mitochondrial swelling and cytochrome c release invitro or in vivo.

Example 14: Compositions of the Present Technology Protect AgainstIschemia-Reperfusion-Induced Myocardial Stunning

Guinea pig hearts will be rapidly isolated, and the aorta will becannulated in situ and perfused in a retrograde fashion with anoxygenated Krebs-Henseleit at constant pressure (40 cm H₂O). Contractileforce will be measured with a small hook inserted into the apex of theleft ventricle and a silk ligature connected to a force-displacementtransducer. Coronary flow will be measured by timing the collection ofpulmonary artery effluent.

Hearts will be perfused with peptide conjugates (1-100 nM);aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) for 30 minutes and then subjected to 30 minutes ofglobal ischemia. Reperfusion will not be performed using perfusionbuffer lacking both peptide conjugates and TBMs (with or withoutaromatic-cationic peptides).

It is anticipated that two-way ANOVA will demonstrate significantdifferences in contractile force, heart rate, and coronary flow inhearts treated with peptide conjugates, aromatic-cationic peptides, orTBMs (with or without aromatic-cationic peptides) compared to untreatedischemic controls. In control hearts, it is anticipated that contractileforce will be significantly lower during the reperfusion period comparedto the pre-ischemic period. In hearts treated with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides), it is anticipated that contractile force during thereperfusion period will be improved compared to untreated controls. Itis further anticipated that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) willprovide complete inhibition of cardiac stunning. In addition, it isanticipated that coronary flow will be well-sustained throughout thereperfusion period and that there will be no decrease in heart rate inhearts treated with peptide conjugates, aromatic-cationic peptides, orTBMs (with or without aromatic-cationic peptides).

It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects with respect totreating or preventing the effects of ischemia-reperfusion inducedmyocardial stunning compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating or preventingthe effects of ischemia-reperfusion induced myocardial stunning.

Example 15: Compositions of the Present Technology Enhance OrganPreservation

For transplantation, the donor hearts are preserved in a cardioplegicsolution during transport. The preservation solution contains highpotassium which effectively stops the heart from beating and conservesenergy. However, the survival time of the isolated heart is quitelimited.

This Example will demonstrate that peptide conjugates, aromatic-cationicpeptides, or TBMs alone or in combination with aromatic-cationicpeptides prolong survival of organs stored for transplant. Isolatedguinea pig hearts will be perfused in a retrograde fashion with anoxygenated Krebs-Henseleit solution at 34° C. After 30 minutes ofstabilization, the hearts will be perfused with a cardioplegic solution(CPS; St. Thomas) alone or in the presence of peptide conjugates (100nM); aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) for 3 minutes. Global ischemia will then be induced bycomplete interruption of coronary flow and maintained for 90 minutes.Reperfusion will be performed for 60 minutes with oxygenatedKrebs-Henseleit solution. Contractile force, heart rate, and coronaryflow will be monitored continuously throughout the procedure.

It is anticipated that the addition of peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) to cardioplegic solution will significantly enhancecontractile function after prolonged ischemia. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for enhancing organpreservation.

Example 16: Compositions of the Present Technology Scavenge HydrogenPeroxide

The effect of peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) on H₂O₂ will be measured byluminol-induced chemiluminescence. Luminol (25 μM) and horseradishperoxidase (0.7 IU) will be added to a solution of H₂O₂ (4.4 nmol)followed by peptide conjugates; aromatic-cationic peptides; or TBMsalone or in combination with aromatic-cationic peptides.Chemiluminescence will be monitored with a Chronolog Model 560aggregometer (Havertown, Pa.) for 20 minutes at 37° C.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) willdose-dependently inhibit the luminol response, demonstrating the abilityto scavenge H₂O₂. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for H₂O₂ scavenging.

Example 17: Compositions of the Present Technology Inhibit LipidPeroxidation

Linoleic acid peroxidation will be induced using the water-solubleinitiator 2,2′-azobis(2-amidinopropane) (ABAP), and lipid peroxidationwill be detected by the formation of conjugated dienes, monitoredspectrophotometrically at 236 nm (E. Longoni, W. A. Pryor, P.Marchiafava, Biochem. Biophys. Res. Commun. 233, 778-780 (1997)).

5 mL of 0.5 M ABAP and varying concentrations of peptide conjugates;aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will be incubated in 2.4 mL linoleic acid suspensionuntil autoxidation rate becomes constant. It is anticipated that peptideconjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will dose-dependently inhibit theperoxidation of linoleic acid.

It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBM in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for inhibiting lipidperoxidation.

Example 18: Compositions of the Present Technology Inhibit LDL Oxidation

Human low density lipoprotein (LDL) will be prepared fresh from storedplasma. LDL oxidation will be induced catalytically by the addition of10 mM Cu₈O₄, and the formation of conjugated dienes will be monitored at234 nm for 5 hours at 37° C. (B. Moosmann and C. Behl, Mol. Pharmacol.61:260-268 (2002).

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) willdose-dependently inhibit the rate of LDL oxidation. It is anticipatedthat administration of peptide conjugates of the present technology willhave synergistic effects in this regard compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for inhibiting LDLoxidation.

Example 19: Compositions of the Present Technology Suppress HydrogenPeroxide Production by Isolated Mouse Liver Mitochondria

This Example will demonstrate the effect of peptide conjugates,aromatic-cationic peptides, or TBMs alone or in combination witharomatic-cationic peptides on H₂O₂ formation in isolated mitochondria.Livers will be harvested from mice, homogenized in ice-cold buffer, andcentrifuged at 13800×g for 10 min. The pellet will be washed once,re-suspended in 0.3 mL of wash buffer, and placed on ice until use. H₂O₂will be measured using luminol chemiluminescence as described previously(Li, et al., Biochim. Biophys. Acta 1428:1-12 (1999). 0.1 mgmitochondrial protein will be added to 0.5 mL potassium phosphate buffer(100 mM, pH 8.0) in the presence of vehicle, peptide conjugates, TBMsalone or in combination with aromatic-cationic peptides, oraromatic-cationic peptides. 25 mM luminol and 0.7 IU horseradishperoxidase will be added, and chemiluminescence will be monitored with aChronolog Model 560 aggregometer (Havertown, Pa.) for 20 minutes at 37°C. The amount of H₂O₂ produced will be quantified as the area under thecurve (AUC) over 20 min, and all data will be normalized to AUC producedby mitochondria alone.

It is anticipated that the amount of H₂O₂ production will besignificantly reduced in the presence of peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for suppressing H₂O₂production in mitochondria.

Example 20: Compositions of the Present Technology SuppressAntimycin-Induced Hydrogen Peroxide Production by Isolated Mouse LiverMitochondria

Livers will be harvested from mice, homogenized in ice-cold buffer, andcentrifuged at 13800×g for 10 min. The pellet will be washed once,re-suspended in 0.3 mL of wash buffer, and placed on ice until use. H₂O₂will be measured using luminol chemiluminescence as described previously(Li, et al., Biochim. Biophys. Acta 1428, 1-12 (1999). 0.1 mgmitochondrial protein will be added to 0.5 mL potassium phosphate buffer(100 mM, pH 8.0) in the presence of vehicle, peptide conjugates,aromatic-cationic peptides, or TBMs with or without aromatic-cationicpeptides. 25 mM luminol and 0.7 IU horseradish peroxidase will be added,and chemiluminescence will be monitored with a Chronolog Model 560aggregometer (Havertown, Pa.) for 20 minutes at 37° C. The amount ofH₂O₂ produced will be quantified as the area under the curve (AUC) over20 min, and all data will be normalized to AUC produced by mitochondriaalone.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) willdose-dependently reduce the spontaneous production of H₂O₂ by isolatedmitochondria. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) willdose-dependently reduce the production of H₂O₂ induced by antimycin inisolated mitochondria. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for suppressingantimycin-induced H₂O₂ production in mitochondria.

Example 21: Compositions of the Present Technology Reduce IntracellularReactive Oxygen Species (ROS) and Increases Cell Survival

To demonstrate that compounds described herein are effective whenapplied to whole cells, neuronal N2A cells will be plated in 96-wellplates at a density of 1×10⁴/well and allowed to grow for 2 days beforetreatment with t-BHP (0.5 or 1 mM) for 40 min. Cells will be washedtwice and incubated in medium alone or medium containing varyingconcentrations of peptide conjugates, aromatic-cationic peptides or TBMswith or without aromatic-cationic peptides for 4 hours. IntracellularROS will be measured using carboxy-H2DCFDA (Molecular Probes, Portland,Oreg., U.S.A.). Cell death will be measured using an MTS cellproliferation assay (Promega, Madison, Wis.).

It is anticipated that incubation with t-BHP will result in adose-dependent increase in intracellular ROS and a decrease in cellviability. It is anticipated that incubation with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will dose-dependently reduce intracellular ROS and increasecell survival with an EC₅₀ in the nM range. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods comprising reducingintracellular ROS levels/production and increasing cell survival.

Example 22: Compositions of the Present Technology Prevent Loss of CellViability

Neuronal N2A and SH-SY5Y cells will be plated in 96-well plate at adensity of 1×10⁴/well and allowed to grow for 2 days before treatmentwith t-butyl hydroperoxide (t-BHP) (0.05-0.1 mM) alone or in thepresence of peptide conjugates, aromatic-cationic peptides or TBMs withor without aromatic-cationic peptides for 24 hours. Cell death will beassessed using an MTS cell proliferation assay (Promega, Madison, Wis.).

It is anticipated that treatment of N2A and SH-SY5Y cells with low dosesof t-BHP (0.05-0.1 mM) for 24 hours will result in a decrease in cellviability. It is anticipated that treatment of cells with peptideconjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will result in a dose-dependent reduction oft-BHP-induced cytotoxicity. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for reducing the loss ofcell viability.

Example 23: Compositions of the Present Technology Decrease CaspaseActivity

N2A cells will be grown on 96-well plates, treated with t-BHP (0.05 mM)in the presence of vehicle, peptide conjugates, aromatic-cationicpeptides, or TBMs with or without aromatic-cationic peptides at 37° C.for 12-24 hours. All treatments will be carried out in quadruplicate.N2A cells will be incubated with t-BHP (50 mM) alone or in the presenceof peptide conjugates, aromatic-cationic peptides, or TBMs with orwithout aromatic-cationic peptides at 37° C. for 12 hours. Cells will begently lifted from the plates with a cell detachment solution (Accutase,Innovative Cell Technologies, Inc., San Diego, Calif., U.S.A.) and willbe washed twice in PBS. Caspase activity will be assayed using a FLICAkit (Immunochemistry Technologies LLC, Bloomington, Minn.). According tothe manufacturer's recommendation, cells will be resuspended (approx.5×10⁶ cells/mL) in PBS and labeled with pan-caspase inhibitorFAM-VAD-FMK for 1 hour at 37° C. under 5% CO₂ while protected fromlight. Cells will then be rinsed to remove the unbound reagent andfixed. Fluorescence intensity in the cells will be measured by a laserscanning cytometer (Beckman-Coulter XL, Beckman Coulter, Inc.,Fullerton, Calif., U.S.A.) using the standard emission filters for green(FL1). For each run, 10,000 individual events will be collected andstored in list-mode files for off-line analysis.

Caspase activation is the initiating trigger of the apoptotic cascade,and it is anticipated that there will be a significant increase incaspase activity after incubation of the cells with 50 mM t-BHP for 12hours, which will be dose-dependently inhibited by peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for decreasing caspaseactivity.

Example 24: Compositions of the Present Technology Inhibit LipidPeroxidation in Cells Exposed to Oxidative Damage

Lipid peroxidation will be evaluated by measuring 4-HNE Michael adducts.4-HNE is one of the major products of the peroxidation of membranepolyunsaturated fatty acids. N2A cells will be seeded on a glass dish 1day before t-BHP treatment (1 mM, 3 hours, 37° C., 5% CO₂) alone or inthe presence of peptide conjugates (10⁻⁸ to 10⁻¹⁰ M), aromatic-cationicpeptides (an equivalent molar dose of aromatic-cationic peptide based onthe concentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group); or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group). Cells will bewashed twice with PBS, fixed 30 minutes with 4% paraformaldehyde in PBSat RT, and washed 3 additional times with PBS. Cells will then bepermeabilized and treated with rabbit anti-HNE antibody followed by asecondary antibody (goat anti-rabbit IgG conjugated to biotin). Cellswill be mounted in Vectashield and imaged using a Zeiss fluorescencemicroscope using an excitation wavelength of 460±20 nm and a longpassfilter of 505 nm for emission.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) will inhibit lipidperoxidation in N2A cells treated with t-BHP. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for inhibiting lipidperoxidation in cells exposed to oxidative damage.

Example 25: Compositions of the Present Technology Inhibit Loss ofMitochondrial Membrane Potential in Cells Exposed to Hydrogen Peroxide

Caco-2 cells will be treated with t-BHP (1 mM) alone or in the presenceof peptide conjugates (0.1 μM); aromatic-cationic peptides (anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group); or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) for 4 hours, andthen incubated with TMRM and examined under CLSM. In cells treated witht-BHP, it is anticipated that TMRM fluorescence will be much reducedcompared to control cells, suggesting generalized mitochondrialdepolarization. In contrast, it is anticipated that treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will protect against mitochondrialdepolarization caused by t-BHP. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for inhibiting the loss ofmitochondrial membrane potential in cells exposed to hydrogen peroxide.

Example 26: Compositions of the Present Technology Prevent Loss ofMitochondrial Membrane Potential and Increased ROS Accumulation in N2ACells Exposed to t-BHP

N2A cells cultured in a glass dish will be treated with 0.1 mM t-BHPalone or in the presence of peptide conjugates (1 nM); aromatic-cationicpeptides (an equivalent molar dose of aromatic-cationic peptide based onthe concentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group) or TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate treatmentgroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate treatment group) for 6 hours. Cells will then beloaded with 10 μM dichlorofluorescin (DCF) (ex/em=485/530) for 30minutes at 37° C., 5% CO₂. Cells will be washed 3 times with HBSS,stained with 20 nM of Mitotracker TMRM (ex/em=550/575 nm) for 15 minutesat 37° C., and examined by confocal laser scanning microscopy.

It is anticipated that the treatment of N2A cells with t-BHP will resultin a loss of TMRM fluorescence, indicating mitochondrial depolarization,and a concomitant increase in DCF fluorescence, indicating an increasein intracellular ROS. It is further anticipated that treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will prevent mitochondrial depolarizationand reduce ROS accumulation. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for inhibiting the loss ofmitochondrial membrane potential and increased ROS accumulation in cellsexposed to t-BHP.

Example 27: Compositions of the Present Technology Prevent ApoptosisCaused by Oxidative Stress

SH-SY5Y cells will be grown in 96-well plates and treated with t-BHP(0.025 mM) alone or in the presence of peptide conjugates,aromatic-cationic peptides, or TBMs with or without aromatic-cationicpeptides at 37° C. for 24 hours. All treatments will be carried out inquadruplicate. Cells will then be stained with 2 mg/mL Hoechst 33342 for20 minutes, fixed with 4% paraformaldehyde, and imaged using a Zeissfluorescent microscope (Axiovert 200M) equipped with the Zeiss Acroplan20× objective. Nuclear morphology will be evaluated using an excitationwavelength of 350±100 m and a longpass filter of 400 nm for emission.All images will be processed and analyzed using MetaMorph software(Universal Imaging Corp., West Chester, Pa., U.S.A.). Uniformly stainednuclei will be scored as healthy, viable neurons. Cells with condensedor fragmented nuclei will be scored as apoptotic. It is anticipated thatpeptide conjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides will prevent SH-SY5Y cell apoptosis inducedby 0.025 mM t-BHP. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing apoptosiscaused by oxidative stress.

Example 28: Compositions of the Present Technology Prevent LipidPeroxidation in Hearts Subjected to Ischemia and Reperfusion

Isolated guinea pig hearts will be perfused in a retrograde manner in aLangendorff apparatus and subjected to various intervals ofischemia-reperfusion. Hearts will be fixed immediately, embedded inparaffin, and sectioned. Immunohistochemical analysis of4-hydroxy-2-nonenol (HNE)-modified proteins will be carried out using ananti-HNE antibody.

It is anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs with or without aromatic-cationicpeptides will prevent lipid peroxidation in hearts subjected to briefintervals of ischemia and reperfusion compared to untreated controls. Itis anticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing lipidperoxidation in organs subjected to ischemia and reperfusion.

Example 29: Compositions of the Present Technology Improve Viability ofIsolated Pancreatic Islet Cells

Islet cells will be isolated from mouse pancreas according to standardprocedures. Peptide conjugates, aromatic-cationic peptides, TBMs with orwithout aromatic-cationic peptides or control vehicle will be added toisolation buffers used throughout the isolation procedure. Mitochondrialmembrane potential will be measured using TMRM (red) and visualized byconfocal microscopy, and apoptosis will be measured by flow cytometryusing annexin V and necrosis by propidium iodide.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) will reduceapoptosis and increase islet cell viability, as measured bymitochondrial membrane potential. It is anticipated that administrationof peptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for improving theviability of isolated pancreatic islet cells.

Example 30: Compositions of the Present Technology Protect AgainstOxidative Damage in Pancreatic Islet Cells

Isolated mouse pancreatic islet cells will be treated with 25 μM t-BHPalone or in the presence of peptide conjugates, aromatic-cationicpeptides, or TBMs with or without aromatic-cationic peptides.Mitochondrial membrane potential will be measured by TMRM (red) andreactive oxygen species will be measured by DCF (green) using confocalmicroscopy. It is anticipated that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) willprotect against oxidative damage in isolated pancreatic islet cells. Itis anticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing oxidativedamage in pancreatic islet cells.

Example 31: Compositions of the Present Technology Protect AgainstParkinson's Disease

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (M_(tox)) is a neurotoxinthat selectively destroys striatal dopaminergic neurons and is anaccepted animal model of Parkinson's Disease.1-methyl-4-phenylpyridinium (MPP⁺), a metabolite of M_(tox), targetsmitochondria, inhibits complex I of the electron transport chain, andincreases ROS production. MPP⁺ is used for in vitro studies becausecells are unable to metabolize M_(tox) to the active metabolite, whileM_(tox) is used for in vivo (i.e., animal) studies.

SN-4741 cells will be treated with buffer; 50 μM MPP⁺; 50 μM MPP⁺ andpeptide conjugates; 50 μM MPP⁺ and aromatic-cationic peptides; or 50 μMMPP⁺ and TBMs; or 50 μM MPP⁺ and TBMs with aromatic-cationic peptidesfor 48 hours. Apoptosis will be measured by fluorescent microscopy withHoechst 33342. It is anticipated that the number of condensed,fragmented nuclei will be significantly increased by MPP⁺ treatment incontrol cells, and that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will reduce the number of apoptotic cells. It is anticipatedthat administration of peptide conjugates of the present technology willhave synergistic effects in this regard compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is further anticipated that peptide conjugates, aromatic-cationicpeptides, or TBMs with or without aromatic-cationic peptides willdose-dependently prevent the loss of dopaminergic neurons in micetreated with M_(tox). Three doses of M_(tox) (10 mg/kg) will be given tomice (n=12) 2 hours apart. Peptide conjugates, aromatic-cationicpeptides, or TBMs with or without aromatic-cationic peptides will beadministered 30 minutes before each M_(tox) injection, and at 1 and 12hours after the last M_(tox) injection. Animals will be sacrificed oneweek later and striatal brain regions will be immunostained for tyrosinehydroxylase activity. Levels of dopamine, DOPAC and HVA levels will bequantified by high pressure liquid chromatography.

It is anticipated that dopamine, DOPAC (3,4 dihydroxyphenylacetic acid)and HVA (homovanillic acid) levels will be significantly reduced byM_(tox) exposure in untreated control mice. It is anticipated thatpeptide conjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides will dose-dependently increase striataldopamine, DOPAC, and HVA levels in mice treated with M_(tox). It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating or preventingParkinson's disease in mammalian subjects.

Example 32: Compositions of the Present Technology Reduce MitochondrialDysfunction in Rats Fed a High-Fat Diet

To determine the potential impact of diet-induced obesity on the controlof cellular redox balance in skeletal muscle, a novel approach tomeasure the rate of mitochondrial H₂O₂ production in permeabilizedskeletal muscle fiber bundles will be developed. See Anderson, et al.,J. Clin. Invest. (doi: 10.1 172/JC137048). During basal (state 4)respiration supported by NADH-linked complex I substrates, the rate ofsuperoxide formation is low, representing 0.1-0.5% of total O₂utilization (Anderson & Neufer, Am. J. Physiol. Cell Physiol. 290:C844-851 (2006); St-Pierre, et al., J. Biol. Chem. 277:44784-44790(2002)). However, respiration supported exclusively by succinate, anFADH-linked complex II substrate, promotes high rates of superoxideproduction by generating reverse electron flow back into complex I(Anderson & Neufer, Am J Physiol Cell Physiol 290:C844-851 (2006);St-Pierre, et al., J. Biol. Chem. 277:44784-44790 (2002); Liu, et al.,J. Neurochem. 80:780-787 (2002); Turrens, et al., Biochem. 1 191:421-427(1980)). This Example describes methods for measuring mitochondrialfunction in permeabilized muscle tissues and examines the effects of ahigh-fat diet on mitochondrial function.

Animals and Reagents.

Thirty male Sprague-Dawley rats will be obtained from Charles RiverLaboratory (Wilmington, Mass.) and housed in a temperature (22° C.) andlight controlled room with free access to food and water. Twenty of theanimals will be maintained on a high (60%) fat diet (Research Dyets,Bethlehem, Pa.). Skeletal muscle will be obtained from anesthetizedanimals (100 mg/kg i.p. ketamine-xylazine). After surgery, animals willbe sacrificed by cervical dislocation while anesthetized. Amplex RedUltra reagent will be obtained from Molecular Probes (Eugene, Oreg.).Stigmatellin and horseradish peroxidase (HRP) will be obtained fromFluka Biochemika (Buchs, Switzerland). All other chemicals will bepurchased from Sigma-Aldrich (St. Louis, Mo.). All animal studies willbe approved by the East Carolina University Institutional Animal Careand Use Committee.

Preparation of Permeabilized Muscle Fiber Bundles.

Briefly, small portions (25 mg) of soleus, red gastrocnemius (RG), andwhite gastrocnemius (WG) muscle will be dissected and placed in ice-coldbuffer X, containing 60 mM K-MES, 35 mM KCl, 7.23 mM K₂EGTA, 2.77 mMCaK₂EGTA, 20 mM imidazole, 0.5 mM DTT, 20 mM taurine, 5.7 mM ATP, 15 mMPCr, and 6.56 mM MgCl₂.6H₂O (pH 7.1, 295 mosmol/kg H₂O). The muscle willbe trimmed of connective tissue and cut down to fiber bundles (2×7 mm,4-8 mg wet wt). Using a pair of needle-tipped forceps under a dissectingmicroscope, fibers will be gently separated from one another to maximizesurface area of the fiber bundle, leaving only small regions of contact.To permeabilize the myofibers, each fiber bundle will be placed inice-cold buffer X containing 50 μg/mL saponin and incubated on a rotatorfor 30 minutes at 4° C. Permeabilized fiber bundles (PmFBs) will bewashed in ice-cold buffer Z containing 110 mM K-MES, 35 mM KCl, 1 mMEGTA, 10 mM K₂HPO₄, 3 mM MgCl₂.6H₂O, 5 mg/mL BSA, 0.1 mM glutamate, and0.05 mM malate (pH 7.4, 295 mOsm), and incubated in buffer Z on arotator at 4° C. until analysis (<2 hours).

Mitochondrial Respiration and H₂O₂ Production Measurements.

High resolution respirometric measurements will be obtained at 30° C. inbuffer Z using the Oroboros O₂K Oxygraph (Innsbruck, Austria).Mitochondrial H₂O₂ production will be measured at 30° C. during state 4respiration in buffer Z (10 μg/mL oligomycin) by continuously monitoringoxidation of Amplex Red using a Spex Fluoromax 3 (Jobin Yvon, Ltd.)spectrofluorometer with temperature control and magnetic stirringat >1000 rpm. Amplex Red reagent reacts with H₂O₂ in a 1:1 stoichiometrycatalyzed by HRP to yield the fluorescent compound resorufin and molarequivalent O₂. Resorufin has excitation/emission characteristics of 563nm/587 nm and is extremely stable once formed. After baselinefluorescence (reactants only) is established, the reaction will beinitiated by addition of a permeabilized fiber bundle to 300 μL ofbuffer Z containing 5 μM Amplex Red and 0.5 U/mL HRP, with succinate at37° C. For the succinate experiments, the fiber bundle will be washedbriefly in buffer Z without substrate to eliminate residual pyruvate andmalate. Where indicated, 10 μg/mL oligomycin will be included in thereaction buffer to block ATP synthase and ensure state 4 respiration. Atthe conclusion of each experiment, PmFBs will be washed indouble-distilled (dd) H₂O to remove salts, and freeze-dried in alyophilizer (LabConco). The rate of respiration will be expressed aspmol per second per mg dry weight, and mitochondrial H₂O₂ productionexpressed as pmol per minute per dry weight.

Statistical Analyses.

Data will be presented as means±SE. Statistical analyses will beperformed using a one-way ANOVA with Student-Newman-Keuls method foranalysis of significance among groups. The level of significance will beset at p<0.05.

It is anticipated that maintaining animals on a 60% fat diet for aperiod of 3 weeks will cause an increase in the maximal rate ofmitochondrial H₂O₂ production. It is anticipated that the addition ofrotenone at the conclusion of succinate titration will eliminate H₂O₂production, confirming complex I as the source of superoxide productionin both control animals and those fed high-fat diets. Mitochondrial H₂O₂production will also be measured by titrating pyruvate/malate in thepresence of antimycin (complex III inhibitor), with the expectation thatanimals fed a high-fat diet will have a higher maximal rate of H₂O₂production than control animals.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs with or without aromatic-cationic peptides will reducemitochondrial dysfunction in mammalian subjects exposed to a high-fatdiet. It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBMs in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for reducing mitochondrialdysfunction in mammalian subjects exposed to a high-fat diet.

Example 33: Compositions of the Present Technology Reduce ROS Productionin Rats Fed a High-Fat Diet

Superoxide production is higher during basal respiration supported byfatty acid versus carbohydrate metabolism, raising the possibility thatthe increase in mitochondrial H₂O₂ production caused by a high-fat dietmay be a result of elevations in cellular H₂O₂ levels (e.g., ROS by aROS-induced ROS release mechanism). To test this hypothesis, the effectsof the peptide conjugates, aromatic-cationic peptides, or TBMs with orwithout aromatic-cationic peptides on mitochondrial function in high-fatfed rats will be examined. Some antioxidants have been shown toeffectively reduce ROS in hearts subjected to myocardial stunning, inpancreatic islet cells after transplantation, and in animal models ofParkinson's disease and amyotrophic lateral sclerosis (Zhao, et al., J.Biol. Chem. 279:34682-34690 (2004); Thomas, et al., J. Am. Soc. Nephr.16, TH-FC067 (2005); Petri, et al., J. Neurochem. 98, 1141-1148 (2006);Szeto, et al., AAPS J. 8: E521-531 (2006)).

Ten rats maintained on a high-fat diet will receive dailyintraperitoneal injections of peptide conjugates (1.5 mg/kg);aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) dissolved in phosphate-buffered saline. Dose responsecurves for peptide conjugates, aromatic-cationic peptides and TBMs withor without aromatic-cationic peptides will be established in vitro andin vivo. Mitochondrial function will be measured according to themethods described herein. It is anticipated that both dose responsecurves will reflect a reduction in mitochondrial H₂O₂ production duringsuccinate-supported respiration.

Next, rats will be placed on a high-fat diet (60%) for six weeks withdaily administration of vehicle, peptide conjugates, aromatic-cationicpeptides, or TBMs with or without aromatic-cationic peptides. It isanticipated that succinate titration experiments conducted onpermeabilized fibers will reveal an increase in the maximal rate of H₂O₂production in high-fat fed rats. It is further anticipated thatpermeabilized fibers from high-fat fed rats will display a higher rateof H₂O₂ production during basal respiration supported bypalmitoyl-carnitine. It is anticipated that high-fat fed rats treatedwith peptide conjugates, aromatic-cationic peptides, or TBMs with orwithout aromatic-cationic peptides, will show a reduction inmitochondrial H₂O₂ production during both succinate andpalmitoyl-carnitine supported respiration. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is further anticipated that basal respiration supported bypyruvate/malate will be slightly increased in fibers from high-fat fedrats, suggesting some degree of uncoupling. However, it is alsoanticipated that in high-fat fed rats, basal rates of pyruvate/malate-or palmitoyl-carnitine-supported respiration will be unaffected by TBM-(with or without aromatic-cationic peptides) or peptideconjugate-treatment, indicating that the normalization of H₂O₂production with TBM- (with or without aromatic-cationic peptides) orpeptide conjugate-treatment is not mediated by an increase in protonleak. It is also anticipated that treatment with TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates will not affect bodyweight gain in high-fat fed rats.

Collectively, these findings will demonstrate that administration of amitochondrial targeted antioxidant, such as the TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology, prevents or compensates for the increase in mitochondrialH₂O₂ production induced by a high-fat diet. As such, the TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates of the presenttechnology are useful in methods for preventing or treating insulinresistance caused by mitochondrial dysfunction in mammalian subjectswith high fat diets.

It is increasingly recognized that the intracellular localization andactivity of many proteins (e.g., receptors, kinases/phosphatases,transcription factors, etc.) is controlled by the oxidation state ofthiol (—SH)-containing residues, suggesting that shifts in theintracellular redox environment can affect a wide variety of cellularfunctions (Schafer and Buetner, Free Radic Biol Med 30, 1191-1212(2001). Glutathione (GSH), the most abundant redox buffer in cells, isreversibly oxidized to GSSG by glutathione peroxidase in the presence ofH₂O₂, and reduced to GSH by glutathione reductase with electrons donatedby NADPH. The ratio of GSH/GSSG is typically very dynamic, and reflectsthe overall redox environment of the cell.

Protein homogenates will be prepared by homogenizing 100 mg of frozenmuscle in a buffer containing 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 2 mMNaOrthovanadate, 2 mM NaPyrophosphate, 5 mM NaF, and protease inhibitorcocktail (Complete), at pH 7.2. After homogenization, 1% Triton X-100will be added to the protein suspension, which will be vortexed andincubated on ice for 5 minutes. Samples will be centrifuged at 10,000rpm for 10 minutes to pellet the insoluble debris. For GSSG measurement,tissue will be homogenized in a solution containing 20 mMMethyl-2-vinylpyridinium triflate to scavenge all reduced thiols in thesample. Total GSH and GSSG will be measured using a commerciallyavailable GSH/GSSG assay (Oxis Research Products, Percipio Biosciences,Foster City, Calif., U.S.A).

It is anticipated that high-fat feeding will cause a reduction in totalcellular glutathione content (GSH_(t)) irrespective of treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides, demonstrating that high-fat intakecompromises GSH-mediated redox buffering capacity in skeletal muscle. Toestablish a link between the increased mitochondrial H₂O₂ productionbrought about by high-fat diet and its effect on overall redoxenvironment of skeletal muscle, both GSH and GSSG will be measured inskeletal muscle from standard chow-fed and high-fat fed rats 1)following a 10 hour fast, and 2) 1 hour after administration of astandard glucose load (oral gavage, 10 hour fasted). In standardchow-fed controls, it is anticipated that glucose ingestion will cause areduction in the GSH/GSSG ratio (normalized to GSH_(t)), presumablyreflecting a shift to a more oxidized state in response to the increasein insulin-stimulated glucose metabolism. In high-fat fed rats, it isanticipated that the GSH/GSSG ratio will be reduced in the 10 hourfasted state relative to standard chow-fed controls and will decreasefurther in response to the glucose ingestion. It is anticipated thattreatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will preserve the GSH/GSSGratio near control levels, even following glucose ingestion. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These findings will demonstrate that a high-fat diet shifts theintracellular redox environment in skeletal muscle to a more oxidizedstate, as compared to controls. It is anticipated that treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides will preserve the intracellular redox statein skeletal muscle, presumably by scavenging primary oxidants, therebycompensating for the reduction in total GSH-mediated redox bufferingcapacity induced by a high-fat diet. Thus, it is anticipated that theadministration of a mitochondrial-targeted antioxidant, such as the TBMs(with or without aromatic-cationic peptides) or peptide conjugates ofthe present technology, will prevent or compensate for the metabolicdysfunction that develops in rats fed a high-fat diet.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for reducing ROSproduction in mammalian subjects exposed to a high-fat diet.

Example 34: Compositions of the Present Technology Prevent InsulinResistance in Rats Fed a High-Fat Diet

To demonstrate that mitochondria-driven changes in the intracellularredox environment may be linked to the etiology of high-fat diet-inducedinsulin resistance, oral glucose tolerance tests will be performed inrats following six weeks of a high-fat diet. On the day of testing, foodwill be removed 10 hours prior to administration of a 2 g/kg glucosesolution via oral gavage. Glucose levels will be determined on wholeblood samples (Lifescan, Milpitas, Calif., U.S.A.). Serum insulin levelswill be determined using a rat/mouse ELISA kit (Linco Research, St.Charles, Mo., U.S.A.). Fasting data will be used to determinehomeostatic model assessment (HOMA)-calculated as fasting insulin(mU/mL)×fasting glucose (mM)/22.5.

Blood glucose and insulin responses to the oral glucose challenge areanticipated to be higher and more sustained in high-fat fed ratscompared with standard chow-fed rats. Treatment of high-fat fed ratswith peptide conjugates, aromatic-cationic peptides, or TBMs with orwithout aromatic-cationic peptides is expected to normalize bloodglucose and insulin responses to the oral glucose challenge. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

It is anticipated that homeostatic model assessment (HOMA) will confirmthe development of insulin resistance in high-fat fed rats, and thattreatment of high-fat fed rats with peptide conjugates,aromatic-cationic peptides, or TBMs with or without aromatic-cationicpeptides will suppress the development of insulin resistance. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

To further assess insulin sensitivity, the phosphorylation state of theinsulin signaling protein Akt in skeletal muscle will be measured 1)following a 10 hour fast, and 2) 1 hour after receiving an oral glucoseload. It is anticipated that in response to glucose ingestion, Aktphosphorylation will increase in skeletal muscle of standard chow-fedcontrols but will remain essentially unchanged in high-fat fed rats,confirming the presence of insulin resistance at the level of insulinsignaling. It is further anticipated that the treatment of high-fat fedrats with peptide conjugates, aromatic-cationic peptides, or TBMs withor without aromatic-cationic peptides will increase Akt phosphorylationin response to glucose ingestion, which, indicates insulin sensitivity.It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBMs in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that administration of a mitochondrial-targetedantioxidant, such as the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, preventsinsulin resistance that develops in rats fed a high-fat diet. As such,the TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods of preventing or treating insulin resistance inmammalian subjects.

Example 35: Compositions of the Present Technology PreventMitochondria-Driven Changes in the Intracellular Redox Environment andInsulin Resistance in Human Subjects

This Example will illustrate the link between mitochondria-drivenchanges in the intracellular redox environment and insulin resistance inhuman subjects.

Mitochondrial H₂O₂ production and respiration in permeabilized skeletalmyofiber bundles from lean, insulin sensitive (BMI=21.6±1.2 kg·m⁻²,HOMA=1.2±0.4), and obese/insulin resistant (BMI=43.0±4.1 kg·m⁻²,HOMA=2.5±0.7) male subjects will be measured. On the day of theexperiment, subjects will report to the laboratory following anovernight fast (approximately 12 hours). A fasting blood sample will beobtained for determination of glucose and insulin. Height and bodyweight will be recorded and skeletal muscle biopsies will be obtainedfrom lateral aspect of vastus lateralis by the percutaneous needlebiopsy technique under local subcutaneous anesthesia (1% lidocaine). Aportion of the biopsy samples will be flash frozen in liquid N2 forprotein analysis, and another portion will be used to preparepermeabilized fiber bundles.

Mitochondrial H₂O₂ production is anticipated to be higher in obesesubjects than in lean subjects in response to titration of succinate,and to be higher during basal respiration supported by fatty acid. BasalO₂ utilization is anticipated to be similar in lean and obese subjects,with the rate of mitochondrial free radical leak higher duringglutamate/malate/succinate and palmitoyl-carnitine supported basalrespiration higher in obese subjects. Finally, it is anticipated thatboth total cellular GSH content and the GSH/GSSG ratio will be lower inthe skeletal muscle of obese subjects, indicating an overall lower redoxbuffer capacity and a more oxidized intracellular redox environment.

These results will show that mitochondrial ROS production and theresulting shift to a more oxidized skeletal muscle redox environment isan underlying cause of high-fat diet-induced insulin resistance. Theanticipated increase in mitochondrial H₂O₂ production is expected to bea primary factor contributing to the shift in overall cellular redoxenvironment. Thus, administration of a mitochondrial-targetedantioxidant, such as the peptide conjugates of the present technology,aromatic-cationic peptides or TBMs with or without aromatic-cationicpeptides is expected to prevent or compensate for the metabolicdysfunction caused by a high-fat diet. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

As such, the TBMs (with or without aromatic-cationic peptides) orpeptide conjugates of the present technology or pharmaceuticallyacceptable salts thereof, such as acetate, tartrate, or trifluoroacetatesalts, are useful in methods for preventing or treating insulinresistance in human subjects.

Example 36: Compositions of the Present Technology in the Prevention andTreatment of Insulin Resistance

To demonstrate the prevention and treatment of insulin resistance, theTBMs (with or without aromatic-cationic peptides) or peptide conjugatesof the present technology will be administered to fatty (fa/fa) Zuckerrats, which are an accepted model of diet-induced insulin resistance. Ascompared to high-fat fed Sprague-Dawley rats (as used in Examples32-34), fatty Zucker rats are anticipated to develop a greater degree ofobesity and insulin resistance under similar conditions. As in Examples32-34, it is anticipated that mitochondrial dysfunction (e.g., increasedH₂O₂ production) will be evident in permeabilized fibers from the Zuckerrats.

To demonstrate the effects of TBMs (with or without aromatic-cationicpeptides) or peptide conjugates on the prevention of insulin resistance,young Zucker rats (˜3-4 weeks of age) will be administered peptideconjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides for approximately 6 weeks. As these youngrats do not yet exhibit signs or symptoms of insulin resistance, theyprovide a useful model for assessing the efficacy of methods ofpreventing insulin resistance. Peptide conjugates (1.0-5.0 mg/kg bodywt); aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will be administered to the rats intraperitoneally(i.p.) or orally (drinking water or oral gavage).

It is predicted that administration of peptide conjugates,aromatic-cationic peptides, or TBM (with or without aromatic-cationicpeptides) will attenuate or prevent the development of whole body andmuscle insulin resistance that normally develops in fatty Zucker rats.Physiological parameters measured will include body weight, fastingglucose/insulin/free fatty acid, oral glucose tolerance (OGTT), in vitromuscle insulin sensitivity (in vitro incubation), biomarkers of insulinsignaling (Akt-P, IRS-P), mitochondrial function studies onpermeabilized fibers (respiration, H₂O₂ production), biomarkers ofintracellular oxidative stress (lipid peroxidation, GSH/GSSG ratio,aconitase activity), and mitochondrial enzyme activity. Control animalswill include wild-type and untreated fatty rats. Successful preventionof insulin resistance by the peptide conjugates of the presenttechnology, aromatic-cationic peptides, or TBM (with or withoutaromatic-cationic peptides) will be indicated by a reduction in one ormore of the markers associated with insulin resistance or mitochondrialdysfunction enumerated above. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBM (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

To demonstrate the effects of the peptide conjugates on treatment ofinsulin resistance, Zucker rats (˜12 weeks of age) will be administeredpeptide conjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides for approximately 6 weeks. As these rats showsigns of obesity and insulin resistance, they will provide a usefulmodel for assessing the efficacy of methods of treating insulinresistance. Peptide conjugates (1.0-5.0 mg/kg body wt);aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will be administered to the rats intraperitoneally(i.p.) or orally (drinking water or oral gavage).

It is predicted that administration of peptide conjugates,aromatic-cationic peptides, or TBMs with or without aromatic-cationicpeptides will reduce the whole body and muscle insulin resistance thatnormally develops in fatty Zucker rats. Parameters measured will includebody weight, fasting glucose/insulin/free fatty acid, oral glucosetolerance (OGTT), in vitro muscle insulin sensitivity (in vitroincubation), biomarkers of insulin signaling (Akt-P, IRS-P),mitochondrial function studies on permeabilized fibers (respiration,H₂O₂ production), biomarkers of intracellular oxidative stress (lipidperoxidation, GSH/GSSG ratio, aconitase activity), and mitochondrialenzyme activity. Controls will include wild-type and untreated fattyrats. Successful treatment of insulin resistance by the peptideconjugates of the present technology, aromatic-cationic peptides, orTBMs with or without aromatic-cationic peptides will be indicated by areduction in one or more of the markers associated with insulinresistance or mitochondrial dysfunction enumerated above. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating or preventinginsulin resistance in mammalian subjects.

Example 37: Compositions of the Present Technology Protect AgainstPrerenal ARI Caused by Ischemia-Reperfusion

This Example will demonstrate the effects of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in protecting a subject from acute renal injury (ARI) causedby ischemia-reperfusion (FR).

Eight Sprague Dawley rats (250-300 g) will be assigned to one of thefollowing groups: (1) sham surgery (no I/R); (2) I/R+saline vehicle; (3)I/R+peptide conjugates; (4) I/R+aromatic-cationic peptides; (5)I/R+TBMs; (6) I/R+TBMs and aromatic-cationic peptides. Peptideconjugates (3 mg/kg in saline); aromatic-cationic peptides (anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group) or TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate treatmentgroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate treatment group) will be administered 30 minutesbefore ischemia and immediately before reperfusion. Control animals willbe given saline alone according to the same schedule.

Rats will be anesthetized with a mixture of ketamine (90 mg/kg, i.p.)and xylazine (4 mg/kg, i.p.). The left renal vascular pedicle will beoccluded using a micro-clamp for 30-45 min. At the end of the ischemicperiod, reperfusion will be established by removing the clamp. At thattime, the contralateral kidney will be removed. After 24 hours ofreperfusion, animals will be sacrificed and blood samples will beobtained by cardiac puncture. Renal function will be determined bymeasuring levels of blood urea nitrogen (BUN) and serum creatinine(BioAssay Systems DIUR-500 and DICT-500).

Renal Morphologic Examination:

Kidneys will be fixed in 10% neutral-buffered formalin and embedded inparaffin wax for sectioning. Three-micron sections will be stained withhematoxylin-eosin (H&E) and periodic acid-Schiff (PAS), and analyzed bylight microscopy. Lesions will be scored based on 1) mitosis andnecrosis of individual cells, 2) necrosis of all cells in adjacentproximal convoluted tubules with survival of surrounding tubules, 3)necrosis confined to the distal third of the proximal convoluted tubulewith a band of necrosis extending across the inner cortex, and 4)necrosis affecting all three segments of the proximal convoluted tubule.

TUNEL Assay for Apoptosis:

Renal tissue sections will be deparaffinized and rehydrated withxylenes, a graded alcohol series, and deionized H₂O, and incubated in 20μg/mL proteinase K for 20 minutes at RT An in situ cell death detectionPOD kit (Roche, Ind., USA) will be used according to the manufacturer'sinstructions. Briefly, endogenous peroxidase activity in the kidneysections will be blocked by incubation for 10 minutes with 0.3% H₂O₂ inmethanol. The sections will be then incubated in a humidified chamber inthe dark for 30 minutes at 37° C. with TUNEL reaction mixture. Afterwashing, the slides will be incubated with 50-100 μL Converter-POD in ahumidified chamber for 30 minutes at RT. The slides will be incubated inDAB solution (1-3 min), counterstained with hemotoxylin, dehydratedthrough a graded series of alcohol, and mounted in Permount formicroscopy.

Immunohistochemistry:

Renal sections will be cut from paraffin blocks and mounted on slides.After removal of paraffin with xylene, the slides will be rehydratedusing graded alcohol series and deionized H₂O. Slides will be heated incitrate buffer (10 mM Citric Acid, 0.05% Tween 20, pH 6.0) for antigenretrieval. Endogenous peroxidase will be blocked with hydrogen peroxide0.3% in methanol. Immunohistochemistry will be then performed using aprimary antibody against heme oxygenase-I (HO-1) (ratanti-HO-1/HMOX1/HSP32 monoclonal antibody (R&D Systems, MN, USA) at1:200 dilution, and secondary antibody (HRP conjugated goat anti-ratIgG, VECTASTAIN ABC (VECTOR Lab Inc. MI, USA)). Substrate reagent3-amino-9-ethylcarbazole (AEC, Sigma, Mo., USA) will be used to developthe slides, with hematoxylin used for counterstaining.

Western Blotting:

Kidney tissue will be homogenized in 2 mL of RIPA lysis buffer (SantaCruz, Calif., USA) on ice and centrifuged at 500×g for 30 minutes toremove cell debris. Aliquots of the supernatants will be stored at −80°C. An aliquot comprising 30 μg of protein from each sample will besuspended in loading buffer, boiled for 5 minutes, and subjected to 10%SDS-PAGE gel electrophoresis. Proteins will be transferred to a PVDFmembrane, blocked in 5% non-fat dry milk with 1% bovine serum albuminfor 1 hour, and incubated with a 1:2000 dilution of anti-HO1/HMOXUHSP32or a 1:1000 diluted anti-AMPKa-1, monoclonal antibody (R&D Systems, MN,USA). Specific binding will be detected using horseradishperoxidase-conjugated secondary antibodies, which will be developedusing Enhanced Chemi Luminescence detection system (Cell Signaling,Mass., USA).

ATP Content Assay:

Immediately following harvesting, kidney tissue will be placed into 10mL 5% trichloroacetic acid with 10 mM DTT, 2 mM EDTA, homogenized onice, incubated on ice for 10 min, centrifuged for 10 minutes at 2000×g,and neutralized with pH 7.6 using 10 N KOH. Following centrifugation for10 minutes at 2000×g, aliquots of the resulting supernatant will bestored at −80° C. ATP will be measured by bioluminescence using acommercially available kit (ATP bioluminescent kit, Sigma, Mo., USA).

Mitochondrial Function:

Renal mitochondria will be isolated and oxygen consumption measured inaccordance with the procedures described herein.

It is anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will improve BUN and serum creatinine values in rats afterischemia and reperfusion compared to untreated ischemic controls, andwill prevent tubular cell apoptosis after ischemia and reperfusion. Itis further anticipated that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) willprevent tubular cell injury after ischemia and reperfusion. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology are effectivein reducing the incidence of ARI caused by ischemia-reperfusion. Theseresults will show that TBMs (with or without aromatic-cationic peptides)or peptide conjugates of the present technology or pharmaceuticallyacceptable salts thereof, such as acetate, tartrate, or trifluoroacetatesalts, are useful in methods for protecting a subject from ARI caused byischemia.

Example 38: Compositions of the Present Technology Protect AgainstPostrenal ARI Caused by Ureteral Obstruction

The effects of the TBMs (with or without aromatic-cationic peptides) orpeptide conjugates of the present technology in protecting a subjectfrom ARI caused by ureteral obstruction will be demonstrated in ananimal model of unilateral ureteral obstruction (UUO).

Sprague-Dawley rats will undergo unilateral ureteral ligation with a 4-0silk suture through a midline abdominal incision under sterileconditions. Ureteral obstruction will be carried out by ligating thelower end of the left ureter, just above the ureterovesical junction.Peptide conjugates (1 mg/kg or 3 mg/kg; n=16); aromatic-cationicpeptides (an equivalent molar dose of aromatic-cationic peptide based onthe concentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group); TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group); TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate treatmentgroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate treatment group); or control vehicle (n=16) will beadministered intraperitoneally, one day prior to UUO and continuing for14 days following UUO.

Renal Histology:

Trichrome sections of paraffin embedded specimens will be examined by aboard-certified pathologist (SVS, renal pathology specialist), andfibrosis scored on a scale of 0-+++.

Immunohistochemical Analysis:

Immunohistochemical staining for macrophages will be carried out using amonoclonal antibody to ED-1 as previously described. Macrophages will becounted in 10 high-power fields (×400) by two independent investigatorsin a blinded fashion. Apoptosis will be measured by TUNEL assay asdescribed in Example 37. The presence of fibroblasts will be examinedusing immunohistochemistry, as described above, using the DAKO #S100-A4antibody (1:100 dilution). Antigen will be retrieved by incubating cellswith Proteinase K for 20 minutes. The remaining immunoperoxidaseprotocol will be carried out according to routine procedures.

It is expected that S100-A4 staining will be present in spindle-shapedinterstitial cells and round, inflammatory cells. Only spindle-shapedcells will be quantified. Staining for 8-OH dG will be done usingProteinase K for antigen retrieval and an antibody provided by the JapanInstitute Control of Aging at a dilution of 1:200-1:500.

Polymerase Chain Reaction Analysis:

Renal expression of heme oxygenase-1 (HO-1) will be measured by RT-PCRaccording to the following: Rat kidneys will be harvested and stored at−80° C. until use. Total RNA will be extracted using the Trizol(R)-Chloroform extraction procedure, and mRNA will be purified using theOligotex mRNA extraction kit (Qiagen, Valencia, Calif., U.S.A.)according to manufacturer instructions. mRNA concentration and puritywill be determined by measuring absorbance at 260 nm. RT-PCR will beperformed using Qiagen One-step PCR kit (Qiagen, Valencia, Calif.,U.S.A.) and an automated thermal cycler (ThermoHybrid, PX2). Thermalcycling will be carried out as follows: initial activation step for 15minutes at 95° C. followed by 35 cycles of denaturation for 45 secondsat 94° C., annealing for 30 seconds at 60° C., extension for 60 secondsat 72° C. Amplification products will be separated on a 2% agarose gelelectrophoresis, visualized by ethidium bromide staining, and quantifiedusing Image J densitometric analysis software. GAPDH will be used as aninternal control.

It is anticipated that the unobstructed contralateral kidneys will showvery little, if any, inflammation or fibrosis in tubules, glomeruli orinterstitium, and that obstructed kidneys of control animals will showmoderate (1-2+) medullary trichrome staining and areas of focalperipelvic 1+ staining. It is anticipated that the cortex will show lessfibrosis than the medulla. It is also anticipated that controlobstructed kidneys will show moderate inflammation, generally scored as1+ in the cortex and 2+ in the medulla. Peptide conjugate-,aromatic-cationic peptide-, or TBM- (with or without aromatic-cationicpeptides) treated obstructed kidneys are expected to show significantlyless trichrome staining, with 0-trace in the cortex and tr-1+ in themedulla. Thus, it is anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will decrease medullary fibrosis in a UUO model. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

Fibroblasts will be visualized by immunoperoxidase forfibroblast-specific protein (FSP-1; aka S100-A4). It is anticipated thatincreased expression of FSP-1 will be found in obstructed kidneys. It isalso anticipated that peptide conjugates (1 mg/kg), aromatic-cationicpeptides (an equivalent molar dose of aromatic-cationic peptide based onthe concentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group), TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group), or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) willsignificantly decrease the amount of fibroblast infiltration inobstructed kidneys. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. Thus, it anticipated that peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will decrease fibroblast expression in a UUO model.

It is anticipated that in untreated kidneys, 2 weeks of UUO will resultin a significant increase in apoptotic tubular cells as compared to thecontralateral kidneys. It is further anticipated that peptide conjugates(1 mg/kg), aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will significantly decrease tubular apoptosis inobstructed kidneys. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. Thus, it is anticipated that peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will decrease tubular apoptosis in a UUO model.

It is anticipated that there will be a significant increase inmacrophage infiltration into obstructed kidneys as compared tocontralateral kidneys after 2 weeks of UUO. It is further expected thattreatment with 1 mg/kg or 3 mg/kg of peptide conjugates,aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup), or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will significantly decrease macrophage infiltration inobstructed kidneys. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. Thus, it is anticipated that peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will decrease macrophage infiltration in a UUO model.

It is anticipated that obstructed kidneys will be associated withincreased proliferation of renal tubular cells, as visualized byimmunoperoxidase for PCNA. It is anticipated that peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will cause a significant decrease in renal tubularproliferation in the obstructed kidneys. It is anticipated that tubularcell proliferation will be decreased by TBMs (with or withoutaromatic-cationic peptides), aromatic-cationic peptides or peptideconjugates at the 1 mg/kg dose, and will be further decreased at the 3mg/kg dose. It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone. Thus, it is anticipated that peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will suppress renal tubular cell proliferation in a UUO model.

It is anticipated that obstructed kidneys will show elevated oxidativedamage compared to contralateral kidneys, as measured by increasedexpression of heme oxygenase-1 (HO-1) and 8-OH dG. It is anticipatedthat treatment with peptide conjugates, aromatic-cationic peptides, orTBMs (with or without aromatic-cationic peptides) will decrease HO-1expression in the obstructed kidney. It is anticipated that 8-OH dGstaining will be detected in both tubular and interstitial compartmentsof the obstructed kidney, that the number of 8-OH dG positive cells willbe significantly increased in obstructed kidneys compared tocontralateral kidneys, and that the number of 8-OH dG positive cellswill be significantly reduced by treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides). It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBMs in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone. Thus, it is anticipated that peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will decrease oxidative damage in a UUO model.

These results will show that peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) areeffective in reducing interstitial fibrosis, tubular apoptosis,macrophage infiltration, and tubular proliferation in an animal model ofARI caused by UUO. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. As such, the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for protecting a subjectfrom ARI caused by ureteral obstruction.

Example 39: Compositions of the Present Technology in the Prevention andTreatment of Contrast-Induced Nephropathy (CIN)

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention and treatment of contrast-inducednephropathy (CIN) in an animal model of ARI.

Animal Model:

A rat model of radiocontrast dye-induced renal failure as described byAgmon, et al., J. Clin. Invest. 94:1069-1075 (1994) will be used. As inhumans, radiocontrast dyes are generally non-toxic when administered toanimals with normal renal function. However, radiocontrast dyes caninduce ARI in animals with impaired renal function. In this model,impaired renal function will be induced by the administration ofindomethacin (10 mg/kg) and L-NAME (10 mg/kg). Animals will be assignedto one of the following groups:

-   -   1. Control (n=8)    -   2. Indomethcin and L-NAME administered 15 minutes apart,        followed by iothalamate (6 mL/kg) (n=7)    -   3. peptide conjugates (3 mg/kg, i.p.) administered 15 minutes        prior to indomethacin/L-NAME/iothalamate administration as        described in Group 2; second dose of peptide conjugates (3        mg/kg) administered immediately after drug exposure (n=9).    -   4. aromatic-cationic peptides (an equivalent molar dose of        aromatic-cationic peptide based on the concentration of the        aromatic-cationic peptide administered in the peptide conjugate        treatment group) administered 15 minutes prior to        indomethacin/L-NAME/iothalamate administration as described in        Group 2; second dose of aromatic-cationic peptides (an        equivalent molar dose of aromatic-cationic peptide based on the        concentration of the aromatic-cationic peptide administered in        the peptide conjugate treatment group) administered immediately        after drug exposure (n=9).    -   5. TBMs (an equivalent molar dose of TBM based on the        concentration of the TBM administered in the peptide conjugate        treatment group) administered 15 minutes prior to        indomethacin/L-NAME/iothalamate administration as described in        Group 2; second dose of TBMs (an equivalent molar dose of TBM        based on the concentration of the TBM administered in the        peptide conjugate treatment group) administered immediately        after drug exposure (n=9).    -   6. TBMs in combination with aromatic-cationic peptides (e.g., an        equivalent molar dose of TBM based on the concentration of TBM        administered in the peptide conjugate treatment group and an        equivalent molar dose of aromatic-cationic peptide based on the        concentration of aromatic-cationic peptide administered in the        peptide conjugate treatment group) administered 15 minutes prior        to indomethacin/L-NAME/iothalamate administration as described        in Group 2; second dose of TBMs in combination with        aromatic-cationic peptides (e.g., an equivalent molar dose of        TBM based on the concentration of TBM administered in the        peptide conjugate treatment group and an equivalent molar dose        of aromatic-cationic peptide based on the concentration of        aromatic-cationic peptide administered in the peptide conjugate        treatment group) administered immediately after drug exposure        (n=9).

Renal Function:

Renal function will be assessed by determining GFR at baseline and 24hours following dye administration. GFR will be determined by creatinineclearance which will be estimated over a 24 hour interval before andafter dye administration. Creatinine clearance will be analyzed bymeasuring plasma and urinary creatinine levels (Bioassay Systems;DICT-500) and urine volume.

Renal Histology:

Kidneys will be fixed in 10% neutral-buffered formalin and embedded inparaffin wax for sectioning. Three-micron sections will be stained withhematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) and analyzed bylight microscopy by a board certified pathologist. Apoptosis will bevisualized by TUNEL labeling.

It is anticipated that control animals will not display a significantdifference in GFR between the first 24 hour period (approx. 235.0±30.5μL/min/g) and the second 24 hour period (approx. 223.7±44.0 μL/min/g).It is anticipated that when contrast dye is administered to animalspre-treated with indomethacin and L-NAME, GFR will decline within 24hours, and that treatment with peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) beforeand after dye administration will reduce the decline in renal function.It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBMs in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

It is anticipated that PAS staining will illustrate normal morphology incontrol kidneys, and a loss of renal brush border and vacuolization incontrast dye-exposed kidneys. It is further anticipated that the defectsin contrast dye-exposed kidneys will be attenuated by treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides). It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone. Thus, it is anticipated thatpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will prevent renal injury in subjectsexposed to radiocontrast dyes.

It is anticipated that control kidneys will show few apoptotic cells,while contrast dye-exposed kidneys will have numerous apoptotic cells.It is further anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will reduce the number of apoptotic cells in contrastdye-exposed kidneys. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology are effectivein reducing renal injury induced by radiocontrast dye exposure. As such,the TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods for treating or preventing acute renal injury causedby contrast dye exposure.

Example 40: Compositions of the Present Technology in the Prevention andTreatment of CIN in Diabetic Subjects

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention and treatment of contrast-inducednephropathy (CIN) in diabetic subjects.

Animal Model:

Impaired renal function caused by diabetes is one of the majorpredisposing factors for contrast induced nephropathy (McCullough, etal., J. Am. Coll. Cardio., 2008, 51, 1419-1428). In this experiment, atotal of 57 Sprague-Dawley rats will be fed a high-fat diet for 6 weeks,followed by the administration of low-dose streptozotocin (30 mg/kg) fora period of 9 weeks. Blood glucose, serum creatinine and Cystatin C willbe measured. Animals meeting the following criteria (n=20) will advanceto CIN studies: Scr>250 μM, Cystatin C >750 ng/mL and bloodglucose >=16.7 μM.

Animals will be administered iohexol and a saline control vehicle;iohexol and peptide conjugates; iohexol and aromatic-cationic peptides;iohexol and TBMs; or iohexol and TBMs+aromatic-cationic peptides.

On day 1, serum samples will be collected and total urine protein willbe measured using a Bradford assay. On days 2 and 3, ˜3 mg/kg peptideconjugates, aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup), TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group), or control vehicle will be administered subcutaneously(s.c.) 30 minutes prior to contrast dye injection (6 mL/kg i.v. tailvein). Peptide conjugate, aromatic-cationic peptide, TBM with or withoutaromatic-cationic peptide or vehicle administration will be repeated at2 and 24 hours post-dye administration. Serum and urine samples will becollected at days 4 and 5. Animals will be euthanized on day 5, and thevital organs harvested. Samples will be analyzed by Student's t-test anddifferences will be considered significant at p<0.05.

Renal Function:

Renal function will be assessed by determining serum and urinarycreatinine at baseline, 48 hours and 72 hours following dyeadministration. The creatinine clearance will be calculated based on theserum and urinary creatinine and urinary volume. Urinary proteinconcentration will be determined by Bradford Protein Assay kit (Sigma,St. Louis, Mo., U.S.A.), and Cystatin C will be measured using a WestangRat Cystatin C kit (Shanghai, P.R.C.).

It is anticipated that control animals will display elevated levels ofserum Cystatin C (an AKI biomarker) and reduced creatinine clearancefollowing contrast dye exposure, and that treatment with peptideconjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides will attenuate these effects. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

Thus, it is anticipated that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology reduce renaldysfunction caused by radiocontrast dye in a diabetic animal model. Assuch, the TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology, or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods for protecting a diabetic subject from acute renalinjury caused by contrast agents.

Example 41: Compositions of the Present Technology in the Prevention andTreatment of CIN in a Glycerol-Induced Rhabdomyolysis Animal Model

This Example demonstrates the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention and treatment of CIN in a glycerol-inducedrhabdomyolysis animal model.

Animal Model:

This Example will utilize animals subjected to glycerol-inducedrhabdomyolysis, as previously described. Parvez, et al., Invest.Radiol., 24:698-702 (1989); Duan, et al., Acta Radiologica,41:503-507(2000). Sprague-Dawley rats with body weight of 300-400 g willbe dehydrated for 24 hours followed by intramuscular (i.m.) injection of25% glycerol solution (v/v) at the dose of 10 mL/kg. Twenty-four hourslater, the animals will be administered a contrast dye with peptideconjugates, aromatic-cationic peptides, TBMs with or withoutaromatic-cationic peptides, or control vehicle according to thefollowing: 1) 25% glycerin+Saline+PBS (n=6), 2) 25%glycerin+diatrizoate+PBS (n=7), 3) 25% glycerin+diatrizoate+peptideconjugates (n=7), 4) 25% glycerin+diatrizoate+aromatic-cationic peptides(n=7), 5) 25% glycerin+diatrizoate+TBMs, and 6) 25%glycerin+diatrizoate+TBMs+aromatic-cationic peptides. The effects ofTBMs (with or without aromatic-cationic peptides) or peptide conjugateson ARI will be demonstrated by comparing the renal functions in animalsfrom each group. Samples will be analyzed by Student's t-test anddifferences will be considered significant at p<0.05.

Renal Function:

Renal function will be assessed by determining serum and urinarycreatinine at baseline, 24 hours after dehydration, and 48 hoursfollowing contrast dye administration. Creatinine clearance will becalculated based on serum and urinary creatinine levels and urinaryvolume. Urinary albumin concentration will be determined using acompetition ELISA assay.

It is anticipated that creatinine clearance will be reduced whencontrast dye is administered to subjects having glycerol-inducedrhabdomyolysis. It is further anticipated that treatment with peptideconjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will attenuate or prevent reduced creatinineclearance. It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBMs in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

Albuminuria is an indicator of increased permeability of the glomerularmembrane, and can result from exposure to contrast dye. It isanticipated that albuminuria will increase when contrast dye isadministered to subjects having glycerol-induced rhabdomyolysis. It isfurther anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will attenuate or prevent albuminuria in such subjects,suggesting that they have a protective effect on the permeability of theglomerular basement membrane in this model. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that PAS staining will illustrate a loss of proximaltubule brush border following administration of contrast dye to subjectshaving glycerol-induced rhabdomyolysis, as well as glomerular swellingand tubular protein cast deposition. It is further anticipated thattreatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will attenuate or preventthese effects in such subjects. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for the prevention andtreatment of CIN in subjects having rhabdomyolysis.

Example 42: Compositions of the Present Technology in the Prevention andTreatment of Nephrotoxicity (CCl₄-induced Chronic Kidney Injury)

This Example demonstrates the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology for the prevention and treatment of carbon tetrachloride(CCl₄)-induced chronic nephrotoxicity.

Animal Model:

Generation of reactive radicals has been implicated in carbontetrachloride-induced nephrotoxicity, in which is characterized by lipidperoxidation and accumulation of dysfunctional proteins. Ozturk, et al.,Urology, 62:353-356 (2003). This Example describes the effect ofadministration of TBMs (with or without aromatic-cationic peptides) orpeptide conjugates for the prevention of carbon tetrachloride(CCl₄)-induced chronic nephrotoxicity.

Study Design and Experimental Protocol:

Sprague-Dawley rats with body weight of 250 g will be fed a 0.35 g/Lphenobarbital solution (Luminal water) for two weeks, and assigned toone of the following groups: 1) luminal water+olive oil,intragastrointestinal (i.g.), 1 mL/kg, twice per week; PBSsubcutaneously (s.c.) 5 days per week; 2) luminal water+50% CCl₄ .i.g.,2 mL/kg, twice per week; and PBS s.c 5 days per week; 3) luminalwater+50% CCl₄ .i.g., 2 mL/kg, twice per week; peptide conjugates (10mg/kg) s.c. 5 days per week; 4) luminal water+50% CCl₄ .i.g., 2 mL/kg,twice per week; aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group) s.c. 5 days per week; 5) luminal water+50% CCl₄ .i.g.,2 mL/kg, twice per week; TBMs (an equivalent molar dose of TBM based onthe concentration of the TBM administered in the peptide conjugatetreatment group) s.c. 5 days per week; 6) luminal water+50% CCl₄ .i.g.,2 mL/kg, twice per week; TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate treatmentgroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate treatment group) s.c. 5 days per week. Trials will runfor a total of 7 weeks.

At the end of fifth week, four subjects from each group will besacrificed for liver histopathological sectioning and fibrosisexamination. At the end of seventh week, all remaining subjects will besacrificed, and kidney and liver tissues harvested for histopathologicalexamination.

Renal Histology:

Kidneys will be fixed in 10% neutral-buffered formalin and embedded inparaffin wax for sectioning. Three-micron sections will be stained withhematoxylin-eosin (H&E) and analyzed by light microscopy by a certifiedpathologist.

It is anticipated that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) will protect renaltubules from CCl₄ nephrotoxicity. H&E staining is anticipated toillustrate that CCl₄ exposure results in tubular epithelial celldegeneration and necrosis. It is also anticipated that animals treatedwith peptide conjugates, aromatic-cationic peptides, or TBMs (with orwithout aromatic-cationic peptides) will show no significanthistopathological changes compared to untreated control animals of Group(1). It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects with respect topreventing or treating CCl₄-induced nephrotoxicity compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

Thus, TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology are useful in methods forpreventing or treating CCl₄-induced nephrotoxicity.

Example 43: Compositions of the Present Technology in the Prevention ofCisplatin-Induced ARI

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology, or pharmaceutically acceptable salts thereof, such asacetate, tartrate, or trifluoroacetate salts, in the prevention ofcisplatin-induced ARI.

Experimental Protocol:

Sprague-Dawley rats (350-400 g) will be given a single dose of cisplatin(7 mg/kg) intraperitoneally (i.p.) on Day 1. Subjects will receivepeptide conjugates (3 mg/kg) (n=8), aromatic-cationic peptides (anequivalent molar dose of aromatic-cationic peptide based on theconcentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group) (n=8), TBMs (an equivalent molar doseof TBM based on the concentration of the TBM administered in the peptideconjugate treatment group) (n=8), TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) (n=8) or salinevehicle (n=8) subcutaneously just prior to cisplatin administration, andonce daily for 3 additional days. Subjects will be placed in metaboliccages for the final 24 hours of the trial for urine collection. At theend of the trial, blood samples will be withdrawn from tail veins andthe kidneys harvested.

Renal Function:

Renal function will be assessed by measuring blood urea nitrogen (BUN),serum creatinine, urine creatinine, and urine protein. GFR will beestimated from creatinine clearance, which will be determined from serumand urinary creatinine, and urinary volume.

Renal Histology:

Kidneys will be fixed in 10% neutral-buffered formalin and embedded inparaffin wax for sectioning. Three-micron sections will be stained withperiodic acid-Schiff (PAS) and analyzed by light microscopy.

It is anticipated that vehicle control subjects will display asignificant reduction in body weight after cisplatin administration, ascompared to body weights prior to cisplatin administration, and thattreatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will attenuate or preventthis effect. It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBMs in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

It is further anticipated that serum creatinine will substantiallyincrease in vehicle control subjects, and that treatment with peptideconjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will attenuate or prevent this effect. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

It is anticipated that vehicle control subjects will display asignificant increase in BUN after cisplatin treatment, and thattreatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will attenuate or preventthis effect. It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBMs in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone. These results will show that TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates protect kidneys fromcisplatin-induced nephropathy.

As such, the TBMs (with or without aromatic-cationic peptides) orpeptide conjugates of the present technology or pharmaceuticallyacceptable salts thereof, such as acetate, tartrate, or trifluoroacetatesalts, are useful in methods for protecting a subject from acute renalinjury caused by cisplatin or similar nephrotoxic agents.

Example 44: Compositions of the Present Technology in the Prevention andTreatment of Acute Liver Failure (ALF)

This Example demonstrates the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention and treatment of acute liver failure (ALF).

Suitable animal models of ALF utilize surgical procedures, toxic liverinjury, or a combination thereof. See Belanger & Butterworth, MetabolicBrain Disease, 20:409-423 (2005). Peptide conjugates, aromatic-cationicpeptides, TBMs with or without aromatic-cationic peptides or controlvehicle will be administered prior to or simultaneously with a toxic orsurgical insult. Hepatic function will be assessed by measuring serumhepatic enzymes (transaminases, alkaline phosphatase), serum bilirubin,serum ammonia, serum glucose, serum lactate, or serum creatinine.Efficacy of the TBMs (with or without aromatic-cationic peptides) orpeptide conjugates of the present technology in preventing ALF will beindicated by a reduction in the occurrence or severity of the ALF asindicated by the above markers, as compared to control subjects.

It is anticipated that toxic or surgical liver insult will cause reducedliver function, and that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will attenuate or prevent these effects. It is anticipatedthat administration of peptide conjugates of the present technology willhave synergistic effects in this regard compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing or treatingALF.

Example 45: Compositions of the Present Technology in the Prevention orTreatment of Hypermetabolism After Burn Injury

Hypermetabolism (HYPM) is a hallmark feature of metabolic disturbanceafter burn injury. Increased energy expenditure (EE) is associated withaccelerated substrate oxidation and shifts of fuel utilization, with anincreased contribution of lipid oxidation to total energy production.Mitochondria dysfunction is closely related to the development of HYPM.This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention and treatment of HYPM.

Sprague Dawley rats will be randomized into the following groups;sham-burn (SB), burn with saline treatment (B), burn with peptideconjugate-treatment (BP), burn with aromatic-cationic peptides (BP2),burn with TBMs (BP3), burn with TBMs+aromatic-cationic peptides (BP4).Catheters will be surgically placed into jugular vein and carotidartery. Band BP, BP2, BP3 and BP4 animals will receive 30% total bodysurface area full thickness burns by immersing the dorsal part into 100°C. water for 12 seconds with immediate fluid resuscitation. BP, BP2, BP3and BP4 animals will receive IV injection of peptide conjugates (2 mg/kgevery 12 hours), aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group every 12 hours), TBMs (an equivalent molar dose of TBMbased on the concentration of the TBM administered in the peptideconjugate treatment group every 12 hours), and TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group every 12 hours)respectively for three days. The EE of the animals will be monitored for12 hours in a TSE Indirect calorimetry System (TSE Co., Germany).

It is anticipated that animals in the B group will show a significantincrease in EE compared to animals in the SB group, and that treatmentwith peptide conjugates, aromatic-cationic peptides, or TBMs (with orwithout aromatic-cationic peptides) will attenuate or prevent thiseffect. It is anticipated that administration of peptide conjugates ofthe present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBMs in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone. These results will show that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) prevents or attenuates burn-induced HYPM.

As such, TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology, or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods for treating burn injuries and secondary complicationsin subjects in need thereof.

Example 46: Compositions of the Present Technology Protect AgainstBurn-Induced Liver Apoptosis

Systemic inflammatory response syndrome (SIRS) and multiple organfailure (MOF) are leading causes of morbidity and mortality in severeburn patients. This Example demonstrates the use of TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates in preventingthese effects.

Six-to-eight week old male C57BL mice will be subjected to 30% totalbody surface (TBSA) burn injury and subsequently injected daily withsaline vehicle; peptide conjugates (5 mg/kg body weight);aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group); TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup); or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group). A weight- and time-matched sham-burn group exposed tolukewarm (˜37° C.) will serve as controls. Liver tissues will becollected 1, 3, and 7 days after burn injury treatment and analyzed forapoptosis (TUNEL), activated caspase levels (Western blot), and caspaseactivity (enzymatic assay).

It is anticipated that burn injury will increase the rate of apoptosisin the liver of burned subjects on all days examined, with the mostdramatic increase predicted to occur on day 7 post-burn injury. It isfurther anticipated that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will attenuate or prevent this effect. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that Western blot analysis will reveal a progressiveincrease in activated caspase-3 following burn injury, as compared tosham control group. It is further anticipated that treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will attenuate or suppress caspase-3activation on days 3 and 7 post-burn, resulting in activated caspase-3levels similar to those of sham control animals. It is anticipated thatthe caspase activity will increase significantly on post-burn day 7, andthe treatment with peptide conjugates, aromatic-cationic peptides, orTBMs (with or without aromatic-cationic peptides) will reduce caspaseactivity to a level not statistically different from that of shamcontrol group. It is further anticipated that there will be a decreasein protein oxidation following burn injury in mice treated with thepeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides), as compared to burn control subjects. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates prevent burn-induced activation ofapoptotic signaling pathways and subsequent liver apoptosis. As such,TBMs (with or without aromatic-cationic peptides) or peptide conjugatesof the present technology or pharmaceutically acceptable salts thereof,such as acetate, tartrate, or trifluoroacetate salts, are useful inmethods for preventing or treating systemic organ damage, such as liverdamage, secondary to a burn.

Example 47: Compositions of the Present Technology in the Prevention ofWound Contraction After Burn Injury

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention of wound contraction.

Burn wounds are typically uneven in depth and severity, with significantareas around coagulated tissue where the injury may be reversible, andinflammatory tissue damage could be prevented. Wound contraction is aprocess which diminishes the size of a full-thickness open wound, andespecially of a full-thickness burn. Tensions developed duringcontraction and the formation of subcutaneous fibrous tissue can resultin tissue deformity, fixed flexure, or fixed extension of a joint (wherethe wound involves an area over the joint). Such complications areespecially relevant in burn healing. No wound contraction will occurwhen there is no injury to the tissue; and maximum contraction willoccur when the burn is full thickness with no viable tissue remaining inthe wound.

Sprague-Dawley rats (male, 300-350 g) will be pre-treated with (1 mg)peptide conjugates administered i.p. (approx. 3 mg/kg) 1 hour prior toburn (65° C. water, 25 seconds, lower back), followed by the topicalapplication of peptide conjugates to the wound (1 mg), and 1 mg peptideconjugates administered i.p. once every 12 hours for 72 hours. Woundswill be observed for up to 3 weeks post-burn. A similar treatmentregimen is followed for the groups treated with aromatic-cationicpeptides (an equivalent molar dose of aromatic-cationic peptide based onthe concentration of the aromatic-cationic peptide administered in thepeptide conjugate treatment group), TBMs (an equivalent molar dose ofTBM based on the concentration of the TBM administered in the peptideconjugate treatment group), or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group).

It is anticipated that the wounds will take on the appearance of a hardscab, which will be quantified as a measure of wound size. It isanticipated that a slower rate of wound contraction will be observed inthe group treated with peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) as compared to burncontrol subjects, such that the burn injury will be less severe in thesesubjects compared to controls. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for treating woundsassociated with a burn injury.

Example 48: Compositions of the Present Technology Alleviate SkeletalMuscle Dysfunction after Burn Injury

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates in the prevention andtreatment of post-burn complications.

It is thought that a major cause of skeletal muscle mitochondrialdysfunction in burns is the result of defects in oxidativephosphorylation (OXPHOS) via stimulation of mitochondrial production ofreactive oxygen species (ROS) and the oxidative damage to themitochondrial DNA (mtDNA). This hypothesis is supported by dataindicating that the ATP synthesis rate significantly decreases and ROSproduction increases in skeletal muscle in response to burn injury. Thisprogression underlies the burn pathophysiology, which includes skeletalmuscle wasting and cachexia.

A clinically relevant murine burn injury model will be used todemonstrate the effects of TBMs (with or without aromatic-cationicpeptides) or peptide conjugates on burn-induced mitochondrialdysfunction and endoplasmic reticulum (ER) stress. The redox state ofthe gastrocnemius muscle immediately below a local cutaneous burn (90°C. for 3 sec) will be evaluated by nitroxide EPR. It is anticipated thatthe redox state in the muscle will be compromised by burn injury, withthe most dramatic effect at 6 hours post-burn.

Peptide conjugates (3 mg/kg), aromatic-cationic peptides (an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofthe aromatic-cationic peptide administered in the peptide conjugatetreatment group), TBMs (an equivalent molar dose of TBM based on theconcentration of the TBM administered in the peptide conjugate treatmentgroup) or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will be administered i.p. 30 minutes before burn, andimmediately after burn. It is anticipated that at the 6-hour time point,treatment with peptide conjugates, aromatic-cationic peptides, or TBMs(with or without aromatic-cationic peptides) will significantly increasethe rate of nitroxide reduction, demonstrating that a decrease inoxidative stress in muscle beneath the burn. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods of preventing or treatingsecondary complications of a burn injury, such as skeletal muscledysfunction.

Example 49: Compositions of the Present Technology Attenuate theProgression of Tissue Damage Following a Burn

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates in the prevention oftissue damage progression following burn injuries. The results will showthat TBMs (with or without aromatic-cationic peptides) or peptideconjugates improve wound healing (i.e., accelerates healing or leads toless scarring) in a partial thickness burn wound.

Sprague Dawley rats will be randomized into the following groups;sham-burn (SB), burn with saline treatment (B), burn with peptideconjugate-treatment (BP), burn with aromatic-cationic peptides (BP2),burn with TBMs (BP3), and burn with TBMs+aromatic-cationic peptides(BP4). Band BP, BP2, BP3 and BP4 animals will receive a 30% total bodysurface area full thickness burns by immersing the dorsal body into 100°C. water for 12 seconds with immediate fluid resuscitation. BP, BP2, BP3and BP4 animals will receive IV injection of peptide conjugates (2 mg/kgevery 12 hours), aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatetreatment group every 12 hours), TBMs (an equivalent molar dose of TBMbased on the concentration of the TBM administered in the peptideconjugate treatment group every 12 hours) and TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group every 12 hours)respectively for three days. Wound re-epithelialization, contraction,and depth will be assessed via gross morphology and histologically overa period of 21 days. For this purpose, immediately after wounding, darkmarks will be applied onto the skin of the animals at the wound edges aswell as 1 cm away from the edges. Wounds will be digitally photographedover 21 days, and image analysis software will be used to measure thearea of the wound (defined as the scab). Distances of the marks from thewound site will be used to assess wound contraction.

At selected time points, wounds will be harvested from the animals.Because the progression from a second to a third degree wound isexpected to occur primarily in the first 48 hours post-burn, sampleswill be harvested at 12, 24, and 48 hours. To monitor the long-termimpact on the wound healing process, samples will be harvested at 2, 7,14, and 21 days. The tissues will be fixed and embedded, and sectionsacross the center of the wounds collected for H&E and trichromestaining.

Apoptosis of hair follicles of the skin will be measured using TUNELlabeling and activated caspase-3 immunostaining using skin samplesobtained between 0 and 48 hours post-burn. Quantification of TUNEL andcaspase-3 staining will be done on digitally acquired images at highpower. The number of positive cells per high power field will bedetermined, and compared among the groups.

Luminescence mapping will be performed using Doppler imaging to assesswound blood flow. Two hours post-burn, the dorsum of the animal will beimaged on a scanning laser Doppler apparatus to quantify the superficialblood flow distribution in the skin within and outside of the burn area.For luminescence mapping, 100 male Sprague-Dawley rats will be used.Eighty animals will receive a large (covering 30% of the total bodysurface area) full-thickness burn injury on the dorsum. This is awell-established model. They will be divided into several groups: onetreated with peptide conjugates, one treated with aromatic-cationicpeptides, one treated with TBMs, one treated with TBMs+aromatic-cationicpeptides and the other with placebo (saline) treatment. Each group willbe further divided into 4 subgroups consisting of 4 time points whereanimals will be sacrificed for further analysis. Prior to sacrifice,luminescence imaging will be carried out, followed by euthanasia andskin tissue sampling for subsequent histology. Another 20 animals willreceive a “sham burn” and will be treated with peptide conjugates,aromatic-cationic peptides, TBMs with or without aromatic-cationicpeptides or saline. Euthanasia will be performed on two animals in eachof the corresponding 4 time points. On average, each animal will behoused for 10 days (including the pre-burn days in the animal farm) inseparate cages.

It is predicted that administration of peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will accelerate wound healing and attenuate the progression ofburn injuries in this model. It is further predicted that treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will reduce burn-induced apoptosis and bloodflow. It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBMs in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for attenuating theprogression of tissue damage following a burn injury, as in theprogression of a partial thickness burn injury to a full-thickness burninjury.

Example 50: Compositions of the Present Technology Protect AgainstSunburn and Attenuates Progression of Tissue Damage Following Sunburn

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates to protect againstsunburn and attenuate the progression of tissue damage following sunburnin a murine model.

Hairless mice, with skin characteristics similar to humans, will beexposed to excessive UV radiation over the course of a week. Subjectswill be randomly divided into the following groups: 1) burn; salinevehicle; 2) burn, peptide conjugates (4 mg/kg per day, low-dose group);3) burn, peptide conjugates (40 mg/kg per day, high-dose group), 4)burn, aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugate low-dosegroup); 5) burn, aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugatehigh-dose group); 6) burn, TBMs (an equivalent molar dose of TBM basedon the concentration of the TBM administered in the peptide conjugatehigh-dose group); 7) burn, TBMs (an equivalent molar dose of TBM basedon the concentration of the TBM administered in the peptide conjugatelow-dose group); 8) burn, TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate high-dosegroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate high-dose group); 9) burn, TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatelow-dose group and an equivalent molar dose of aromatic-cationic peptidebased on the concentration of aromatic-cationic peptide administered inthe peptide conjugate low-dose group). Peptide conjugates,aromatic-cationic peptides, or TBMs with or without aromatic-cationicpeptides will be administered intravenously twice per day for sevendays. Parameters measured will include wound contraction,re-epithelialization distance, cellularity, and collagen organization.Ki67 proliferation antigen will be assessed, as well as TUNEL andcaspase-3 activation. Blood flow will be measured by luminescencemapping.

It is predicted that administration of peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will accelerate wound healing and attenuate the progression ofsunburn injuries in this model. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for protecting againstsunburn and attenuating the progression of tissue damage followingsunburn.

Example 51: Compositions of the Present Technology AttenuateBurn-Induced Hypermetabolism by Down-Regulating UCP-1 Expression inBrown Adipose Tissue

Hypermetabolism is the hallmark feature of metabolic disturbance afterburn injury. Mitochondrial dysfunction occurs after burns, and isclosely related to the development of hypermetabolism (and alteredsubstrate oxidation). Uncoupling protein 1 (UCP-1) is expressed in thebrown adipose tissue, and plays a key role in producing heat. ThisExample will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology down-regulateUCP-1 expression following burn injury.

Methods.

Sprague Dawley rats will be randomly divided into the following groups;sham (S), sham with saline vehicle (SSal), sham with peptideconjugate-treatment (SC), sham with aromatic-cationic peptides (SC2),sham with TBMs (SC3), sham with TBMs+aromatic-cationic peptides (SC4),burn with saline vehicle (BSal), burn with peptide conjugate-treatment(BC), burn with aromatic-cationic peptides (BC2), burn with TBMs (BC3)and burn with TBMs+aromatic-cationic peptides (BC4). The dorsal aspectof burn subjects will be immersed into 100° C. water for 12 seconds toproduce third degree 30% TBSA burns under general anesthesia. Sham burnwill be produced by immersion in lukewarm water. Subjects will receive40 mL/kg intraperitoneal saline injection for the resuscitationfollowing the injury. A venous catheter will be placed surgically intothe right jugular vein subsequent to sham or burn injury. Peptideconjugates (2 mg/kg), aromatic-cationic peptides (an equivalent molardose of aromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugate group),TBMs (an equivalent molar dose of TBM based on the concentration of theTBM administered in the peptide conjugate group), TBMs in combinationwith aromatic-cationic peptides (e.g., an equivalent molar dose of TBMbased on the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) or saline vehiclewill be infused for 7 days (4 mg/kg/day) using osmotic pumps (Durect,Calif.). Indirect calorimetry will be performed for 24 hours at 6 daysafter burn injury in a TSE Indirect calorimetry System (TSE Co.,Germany), and VO2, VCO2 and energy expenditure will be recorded everysix minutes. Interscapullar brown adipose tissue will be collected afterthe indirect calorimetry, and UCP-1 expression in the brown adiposetissue will be evaluated by Western blot.

It is anticipated that VO2, VCO2, and energy expenditure will besignificantly increased in the BSal group, as compared to the SSalgroup, and that treatment with peptide conjugates, aromatic-cationicpeptides, or TBMs (with or without aromatic-cationic peptides) willsignificantly attenuate this effect. It is further anticipated thatUCP-1 expression in the BSal group will be higher than in the SSalgroup, with UCP-1 levels in the BC, BC2, BC3 and BC4 groups lower thanin the BSal group. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates attenuate burn-induced hypermetabolismby the down regulation of UCP-1 expression in brown adipose tissue. Assuch, the TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology, or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods for treating a subject suffering from a burn injury.

Example 52: Compositions of the Present Technology Induce ATP SynthesisFollowing a Burn Injury

This Example will demonstrate that TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates increase the rate ofATP synthesis following a burn injury using ³¹P NMR and electronparamagnetic resonance (EPR) in vivo.

It is thought that a major cause of skeletal muscle mitochondrialdysfunction in burns is the result of defects in oxidativephosphorylation (OXPHOS) via stimulation of mitochondrial production ofreactive oxygen species (ROS) and the oxidative damage to themitochondrial DNA (mtDNA). This hypothesis is supported by dataindicating that the ATP synthesis rate significantly decreases and ROSproduction increases in skeletal muscle in response to burn injury. Thisprogression underlies the burn pathophysiology, which includes skeletalmuscle wasting and cachexia.

Material and Methods.

Male 6-week-old CD1 mice weighing 20-25 g will be anesthetized byintraperitoneal (i.p.) injection of 40 mg/kg pentobarbital sodium. Theleft hind limb of all mice in all groups will be shaved. Burn subjectswill be subjected to a nonlethal scald injury of 3-5% total body surfacearea (TBSA) by immersing the left hind limb in 90° C. water for 3seconds.

NMR spectroscopy is described in detail in Padfield, et al., Proc. Natl.Acad. Sci., 102:5368-5373 (2005). Briefly, mice will be randomizedinto 1) burn+control vehicle, 2) burn+peptide conjugate, 3)non-burn+control vehicle, 4) non-burn+peptide conjugate, 5)burn+aromatic-cationic peptides, 6) non-burn+aromatic-cationic peptides,7) burn+TBM, 8) non-burn+TBM, 9) burn+TBM+aromatic-cationic peptides,and 10) non-burn+TBM+aromatic-cationic peptides groups. The peptideconjugates (3 mg/kg), aromatic-cationic peptides (an equivalent molardose of aromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugate group),TBMs (an equivalent molar dose of TBM based on the concentration of theTBM administered in the peptide conjugate group), or TBMs in combinationwith aromatic-cationic peptides (e.g., an equivalent molar dose of TBMbased on the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) will be injectedintraperitoneally 30 minutes prior to the burn and immediately after theburn. NMR experiments will be performed in a horizontal bore magnet(proton frequency 400 MHz, 21 cm diameter, Magnex Scientific) using aBruker Avanee console. A 90° pulse will be optimized for detection ofphosphorus spectra (repetition time 2 s, 400 averages, 4K data points).Saturation 90°-selective pulse trains (duration 36.534 ms, bandwidth 75Hz) followed by crushing gradients will be used to saturate the γ-ATPpeak. The same saturation pulse train will be also applied downfield ofthe inorganic phosphate (Pi) resonance, symmetrically to the γ-ATPresonance. T1 relaxation times of Pi and phosphocreatine (PCr) will bemeasured using an inversion recovery pulse sequence in the presence ofγ-ATP saturation. An adiabatic pulse (400 scans, sweep with 10 KHz, 4Kdata) will be used to invert Pi and PCr, with an inversion time between152 ms and 7651 ms.

EPR spectroscopy is described in detail in Khan, et al., Mol. Med. Rep.1:813-819 (2008). Briefly, mice will be randomized into 1) burn+controlvehicle, 2) burn+peptide conjugate, 3) non-burn+control vehicle, 4)non-burn+peptide conjugate, 5) burn+aromatic-cationic peptide, 6)non-burn+aromatic-cationic peptides, 7) burn+TBM, 8) non-burn+TBM, 9)burn+TBM+aromatic-cationic peptides, and 10)non-burn+TBM+aromatic-cationic peptides groups. The peptide conjugates(3 mg/kg), aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on the concentration of thearomatic-cationic peptide administered in the peptide conjugate group),TBMs (an equivalent molar dose of TBM based on the concentration of theTBM administered in the peptide conjugate group), or TBMs in combinationwith aromatic-cationic peptides (e.g., an equivalent molar dose of TBMbased on the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) will be injectedintraperitoneally at 0, 3, 6, 24, and 48 hours post-burn. EPRmeasurements will be carried out with an I.2-GHz EPR spectrometerequipped with a microwave bridge and external loop resonator designedfor in vivo experiments. The optimal spectrometer parameters will be:incident microwave power, 10 mW; magnetic field center, 400 gauss;modulation frequency, 27 kHz. The decay kinetics ofintravenously-injected nitroxide (150 mg/kg) will be measured at thevarious time points, to assess the mitochondrial redox status of themuscle.

It is anticipated that control subjects will display a significantlyelevated redox status after a burn injury, and a significant reductionof the ATP synthesis rate. It is further anticipated that treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will induce a significant increase in theATP synthesis rate in burned mice, as compared to burn controls. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBMs in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone.

These results will show that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) induces ATP synthesis rate possibly via a recovery of themitochondrial redox status or via the peroxisome proliferator activatedreceptor-gamma coactivator-1β (PGC-1β). It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone. Thus, it is predicted that themitochondrial dysfunction caused by burn injury is attenuated byadministration of the peptide conjugates, aromatic-cationic peptides, orTBMs (with or without aromatic-cationic peptides).

It is also predicted that administration of the peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will increase ATP synthesis rate substantially even in controlhealthy mice. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods of preventing or treatingsecondary complications of a burn injury, such as skeletal muscledysfunction.

Example 53: Compositions of the Present Technology Reduce MitochondrialAconitase Activity in Burn Injury

Mitochondrial aconitase is part of the TCA cycle and its activity hasbeen directly correlated with the TCA flux. Moreover, its activity isinhibited by ROS, such that it is considered an index of oxidativestress. This Example will demonstrate the effects of TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates of the presenttechnology on mitochondrial aconitase activity.

Murine subjects will be subjected to burn injury or sham andadministered peptide conjugates, aromatic-cationic peptides, TBMs aloneor in combination with aromatic-cationic peptides, or control vehicle asdescribed above. Mitochondria will be isolated from burned and controltissues and mitochondrial aconitase activity assessed using acommercially available kit.

It is anticipated that mitochondrial aconitase activity will beincreased in both burned (local burn effect) and contralateral to burnedleg (systemic burn effect), most probably due to the hypermetabolisminduced by burn injury. Thus, the increased ROS production known tooccur in burn injury, which could inhibit mitochondrial aconitaseactivity, will likely not overcome the hypermetabolic effect withrespect to mitochondrial aconitase activity and TCA flux. A similarresult has been also shown in the case of exercise/repeated contractionsin intact human and isolated mouse skeletal muscle, although an increasein ROS is also observed in this situation.

Thus, it is further anticipated that treatment with peptide conjugates,aromatic-cationic peptides or TBMs (with or without aromatic-cationicpeptides) will reduce mitochondrial aconitase activity to sham controllevels in subjects receiving a burn injury. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for reducing mitochondrialaconitase activity following a burn injury.

Example 54: Compositions of the Present Technology in the Prevention orTreatment of Metabolic Syndrome

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the prevention and treatment of Metabolic Syndrome.

Sprague Dawley rats will be fed with a high-fat diet (HFD) for 6 weeksand then administered a single dose of STZ (30 mg/kg). The rats will bemaintained on HFD until 14 weeks after STZ administration. Controlsubjects fed normal rat chow (NRC) for 6 weeks will be administeredcitrate buffer without STZ. After 5 months, diabetic subjects will betreated with peptide conjugates (10 mg/kg, 3 mg/kg, or 1 mg/kg s.c.q.d.(subcutaneously, once daily)), aromatic-cationic peptides (an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofthe aromatic-cationic peptide administered in the peptide conjugategroup), TBMs (an equivalent molar dose of TBM based on the concentrationof the TBM administered in the peptide conjugate group), TBMs incombination with aromatic-cationic peptides (e.g., an equivalent molardose of TBM based on the concentration of TBM administered in thepeptide conjugate treatment group and an equivalent molar dose ofaromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) or control vehicle (saline) 5 days per week for 10weeks. The study groups will be as follows:

Group A: HFD/STZ+peptide conjugates 10 mg/kg s.c.q.d. (Mon-Fri.), n=12;

Group B: HFD/STZ+peptide conjugates 3 mg/kg s.c.q.d. (Mon-Fri.), n=12;

Group C: HFD/STZ+peptide conjugates 1 mg/kg s.c.q.d. (Mon-Fri.), n=10;

Group D: HFD/STZ+control vehicle s.c.q.d. (Mon-Fri.), n=10;

Group E: NRC+control vehicle s.c.q.d. (Mon-Fri.), n=10;

Group F: HFD/STZ+aromatic-cationic peptide (an equivalent molar dose ofaromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 10 mg/kg s.c.q.d. peptideconjugate group), n=12

Group G: HFD/STZ+aromatic-cationic peptide (an equivalent molar dose ofaromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 3 mg/kg s.c.q.d. peptideconjugate group), n=12

Group H: HFD/STZ+aromatic-cationic peptide (an equivalent molar dose ofaromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 1 mg/kg s.c.q.d. peptideconjugate group), n=12

Group I: HFD/STZ+TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the 10 mg/kg s.c.q.d. peptideconjugate group), n=12

Group J: HFD/STZ+TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the 3 mg/kg s.c.q.d. peptideconjugate group), n=12

Group K: HFD/STZ+TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the 1 mg/kg s.c.q.d. peptideconjugate group), n=12.

Group L: HFD/STZ+TBMs in combination with aromatic-cationic peptides(e.g., an equivalent molar dose of TBM based on the concentration of TBMadministered in the 10 mg/kg s.c.q.d. peptide conjugate treatment groupand an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 10 mg/kgs.c.q.d. peptide conjugate treatment group), n=12

Group M: HFD/STZ+TBMs in combination with aromatic-cationic peptides(e.g., an equivalent molar dose of TBM based on the concentration of TBMadministered in the 3 mg/kg s.c.q.d. peptide conjugate treatment groupand an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 3 mg/kgs.c.q.d. peptide conjugate treatment group), n=12

Group N: HFD/STZ+TBMs in combination with aromatic-cationic peptides(e.g., an equivalent molar dose of TBM based on the concentration of TBMadministered in the 1 mg/kg s.c.q.d. peptide conjugate treatment groupand an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 1 mg/kgs.c.q.d. peptide conjugate treatment group), n=12

It is anticipated that HFD feeding for 6 weeks will produce obvious bodyweight gain, and that STZ administration will increase blood glucose andhyperlipidemia, indicating a metabolic syndrome-like disorder in thesesubjects. Hence, the protocol will have induced metabolic syndrome inthese subjects.

During the 10-week period of treatment, no obvious changes in bodyweight or blood glucose level are expected in subjects receiving peptideconjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides). The blood glucose of NRC group is expectedto stay in normal range, while that of STZ treatment groups is predictedto remain higher than throughout the 10-week period trial period.

It is anticipated that the blood triglyceride level of HFD/STZ rats willbe much higher than in NRC rats before treatment with peptideconjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides), and will be reduced to normal levelsfollowing 10 weeks of peptide conjugate-, aromatic-cationic peptide- orTBM- (with or without aromatic-cationic peptides) administration,demonstrating beneficial effects on lipid metabolism. It is anticipatedthat administration of peptide conjugates of the present technology willhave synergistic effects in this regard compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing or treatingmetabolic syndrome.

Example 55: Compositions of the Present Technology Prevent HighGlucose-Induced Injury to Human Retinal Epithelial Cells

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates for the prevention ofhigh glucose-induced injury to human retinal epithelial cells (HREC).

Methods of HREC culture useful in the studies of the present technologyare known. See generally, Li, et al., Clin. Ophthal. Res. 23:20-2(2005); Premanand, et al., Invest. Ophthalmol. Vis. Sci. 47:2179-84(2006). Briefly, HREC cells will be cultured under one of theseconditions: 1) normal control; 2) 30 mM glucose; 3) 30 mMglucose+peptide conjugates; 4) 30 mM glucose+an aromatic-cationicpeptide; 5) 30 mM glucose+TBM; 6) 30 mM glucose+TBM+aromatic-cationicpeptides. Survival of HRECs in high glucose co-treated with variousconcentrations of peptide conjugates (10 nM, 100 nM, 1 μM, 10 μM) willbe measured by flow cytometry using Annexin V. See generally, Koopman,et al., Blood 84:1415 (1994); Homburg, et al., Blood X5: 532 (1995);Vermes, et al. J. Immunol. Meth. 184:39 (1995); Fadok, et al., J.Immunol. 148:2207 (1992).

The survival of HRECs in high glucose co-treated with peptideconjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides will be tested at 24 hours and 48 hours. Itis predicted that survival of HRECs will be significantly improved withthe administration of peptide conjugates, aromatic-cationic peptides, orTBMs (with or without aromatic-cationic peptides) as compared tocontrols, with a reduction in apoptotic and necrotic cells. Treatmentwith peptide conjugates, aromatic-cationic peptides, or TBMs (with orwithout aromatic-cationic peptides) is also anticipated to reduce theproduction of ROS. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

To demonstrate that a mitochondrial-mediated pathway will be importantin TBM-(with or without aromatic-cationic peptides) or peptideconjugate-mediated protection against high glucose-induced cell death,mitochondrial membrane potential will be measured by flow cytometryusing TMRM. It is anticipated that incubating the HRECs withhigh-glucose for 24 or 48 hours will lead to a rapid loss ofmitochondrial membrane potential, and that concurrent treatment withpeptide conjugates, aromatic-cationic peptides, or TBMs (with or withoutaromatic-cationic peptides) will prevent or attenuate this effect. Theseresults will show that peptide conjugates, aromatic-cationic peptides,or TBMs (with or without aromatic-cationic peptides) prevent themitochondrial membrane potential loss caused by exposure to a highglucose environment. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

It is expected that glucose (30 mmol/L) will induce cytochrome c releasefrom the mitochondria of HRECs. Fixed HRECs will be immunolabeled with acytochrome c antibody and a mitochondrial specific protein antibody(HSP60). It is predicted that confocal microscopic analysis will showthat HRECs in normal culture and in cultures containing peptideconjugates, aromatic-cationic peptides, or TBMs with or withoutaromatic-cationic peptides co-treated with glucose have overlappingcytochrome c staining and mitochondria staining, indicatingcolocalization of cytochrome c and mitochondria. It is anticipated thatafter treatment with 30 mmol/L glucose for 24 or 48 hours, cytochrome cwill be observed in the cytoplasm of HRECs, indicating that glucoseinduces the release of cytochrome c from the mitochondria to cytoplasmin HREC cells, and that treatment with peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will prevent or attenuate this effect. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates promote the survival of HREC cells in ahigh glucose environment. As such, the TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology, or pharmaceutically acceptable salts thereof, such asacetate, tartrate, or trifluoroacetate salts, are useful in methods forthe prevention of diabetic retinopathy.

Example 56: Compositions of the Present Technology Prevent DiabeticRetinopathy in Rats Fed a High-Fat Diet

This Example will demonstrate use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates in the prevention ofdiabetic retinopathy in rats fed a high-fat diet (HFD).

A rat model of diabetes will be established by combination of 6-week HFDand either 1) a low-dose STZ (30 mg/kg) injection, or 2) a single highdose of STZ (65 mg/kg) in Sprague-Dawley rats. See generally,Srinivasan, et al., Pharm. Res. 52(4):313-320 (2005). Controls will bemaintained on normal rat chow (NRC). Treatment groups will be asfollows:

Group A: 12 HFD/STZ peptide conjugates 10 mg/kg s.c

Group B: 12 HFD/STZ peptide conjugates 3 mg/kg s.c.

Group C: 12 HFD/STZ peptide conjugates 1 mg/kg s.c.

Group D: 10 HFD/STZ control vehicle. s.c.

Group E: 10 NRC control vehicle. s.c.

Group F: 12 HFD/STZ aromatic-cationic peptide (an equivalent molar doseof aromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 10 mg/kg s.c.q.d. peptideconjugate group)

Group G: 12 HFD/STZ aromatic-cationic peptide (an equivalent molar doseof aromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 3 mg/kg s.c.q.d. peptideconjugate group)

Group H: 12 HFD/STZ aromatic-cationic peptide (an equivalent molar doseof aromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 1 mg/kg s.c.q.d. peptideconjugate group)

Group I: 12 HFD/STZ TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the 10 mg/kg s.c.q.d. peptideconjugate group)

Group J: 12 HFD/STZ TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the 3 mg/kg s.c.q.d. peptideconjugate group)

Group K: 12 HFD/STZ TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the 1 mg/kg s.c.q.d. peptideconjugate group).

Group L: TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the 10 mg/kg s.c.q.d. peptide conjugate treatment groupand an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 10 mg/kgs.c.q.d. peptide conjugate treatment group)

Group M: TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the 3 mg/kg s.c.q.d. peptide conjugate treatment groupand an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 3 mg/kgs.c.q.d. peptide conjugate treatment group)

Group N: TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the 1 mg/kg s.c.q.d. peptide conjugate treatment groupand an equivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 1 mg/kgs.c.q.d. peptide conjugate treatment group)

Eyes will be harvested and subjects assessed for cataract formation,epithelial changes, integrity of the blood-retinal barrier, retinalmicrovascular structure, and retinal tight junction structure usingmethods known in the art.

It is anticipated that administration of peptide conjugates,aromatic-cationic peptides, or TBMs (with or without aromatic-cationicpeptides) will result in a prevention or reversal of cataract formationin the lenses of diabetic rats. It is further anticipated thatadministration of peptide conjugates, aromatic-cationic peptides or TBMs(with or without aromatic-cationic peptides) will reduce epithelialcellular changes in both STZ rat model and HFD/STZ rat model, and resultin improved inner blood-retinal barrier function compared to controlsubjects. It is anticipated that administration of peptide conjugates ofthe present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

It is anticipated that administration of peptide conjugates,aromatic-cationic peptides or TBMs (with or without aromatic-cationicpeptides) will reduce retinal microvascular changes observed in STZ orHFD/STZ rats. It is further anticipated that the tight junctions, asvisualized by claudin-5 localization, will be uniformly distributedalong the retinal vessels in control subjects, and non-uniformly inHFD/STZ subjects. It is further anticipated that treatment with peptideconjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides) will prevent, reverse, or attenuate thiseffect. It is anticipated that administration of peptide conjugates ofthe present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBMs in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

These results will collectively establish that peptide conjugates,aromatic-cationic peptides or TBMs (with or without aromatic-cationicpeptides) prevent/compensate for the negative effects of diabetes in theeye, e.g., cataracts and microvasculature damage. As such, the TBMs(with or without aromatic-cationic peptides) or peptide conjugates ofthe present technology, or pharmaceutically acceptable salts thereof,such as acetate, tartrate, or trifluoroacetate salts, are useful inmethods for preventing or treating ophthalmic conditions associated withdiabetes in human subjects.

Example 57: Compositions of the Present Technology in the Prevention andTreatment of Heart Failure

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates in the prevention andtreatment of hypertensive cardiomyopathy and heart failure. This Examplewill further demonstrate the role of NADPH and mitochondria inangiotensin II (Ang II)-induced cardiomyopathy, and in cardiomyopathicmice overexpressing the α subunit of the heterotrimeric Gq protein(Gαq).

Ventricles from mouse neonates younger than 72 hours will be dissected,minced, and enzymatically digested with Blendzyme 4 (45 mg/mL, Roche).After enzymatic digestion, cardiomyocytes will be enriched usingdifferential pre-plating for 2 hours, and seeded on fibronectin-coatedculture dishes for 24 hours in DMEM (Gibco) with 20% Fetal Bovine Serum(Sigma) and 25 μM Arabinosylcytosine (Sigma). Cardiomyocytes will bestimulated with Angiotensin II (1 μM) for 3 hours in serum-free DMEMcontaining 0.5% insulin transferrin-selenium (Sigma), 2 mM glutamine,and 1 mg/mL BSA. Cardiomyocytes are simultaneously treated with one ofthe following: peptide conjugates (1 nM), aromatic-cationic peptides (anequivalent molar dose of aromatic-cationic peptide based onconcentration of the aromatic-cationic peptide administered in thepeptide conjugate group), TBMs (an equivalent molar dose of TBM based onconcentration of the TBM administered in the peptide conjugate group),TBMs in combination with aromatic-cationic peptides (e.g., an equivalentmolar dose of TBM based on the concentration of TBM administered in thepeptide conjugate treatment group and an equivalent molar dose ofaromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group), N-acetyl cysteine (NAC: 0.5 mM), or PBS control. Tomeasure mitochondrial superoxide concentration, Mitosox (5 pM) will beincubated for 30 minutes at 37° C. to load cardiomyocytes, followed by 2washes with Hanks Balanced Salt Solution. Samples will be analyzed usingexcitation/emission of 488/625 nm by flow cytometry. Flow data will beanalyzed using FCS Express (De Novo Software, Los Angeles, Calif.,U.S.A.), and presented as histogram distributions of Mitosoxfluorescence intensity.

Mouse Experiments, Drug Delivery, Echocardiography and Blood PressureMeasurement.

Six to ten mice will be included in each experimental group (Saline, AngII, Ang II+peptide conjugate, Ang II+aromatic-cationic peptide, AngII+TBM, Ang II+TBM+aromatic-cationic peptide, WT, Gαq, Gαq+peptideconjugate, Gαq+aromatic-cationic peptide, Gαq+TBM,Gαq+TBM+aromatic-cationic peptide). A pressor dose of Ang II (1.1mg/kg/d) will be continuously administered for 4 weeks usingsubcutaneous Alzet 1004 osmotic minipumps, either alone or in thepresence of with peptide conjugates (3 mg/kg/d), aromatic-cationicpeptides (an equivalent molar dose of aromatic-cationic peptide based onconcentration of the aromatic-cationic peptide administered in thepeptide conjugate group), TBMs (an equivalent molar dose of TBM based onconcentration of the TBM administered in the peptide conjugate group),or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group). Echocardiography will be performed at baseline and 4weeks after pump implantation using a Siemens Acuson CV-70 equipped witha 13 MHz probe. Under 0.5% isoflurane to reduce agitation, standardM-mode, conventional and Tissue Doppler images will be taken, andfunctional calculations will be performed according to American Societyof Echocardiography guidelines. MTI will be calculated as the ratio ofthe sum of isovolemic contraction and relaxation time to LV ejectiontime. An increase in MPI is an indication that a greater fraction ofsystole is spent to cope with the pressure changes during the isovolemicphases. As a reference for the effect of the TBM (with or withoutaromatic-cationic peptides) or peptide conjugate in Ang II treated mice,a genetic mouse model of Rosa-26 inducible-mCAT will be included, inwhich mitochondrial catalase will be overexpressed for two weeks beforeAng II treatment.

Blood pressure will be measured in a separate group of mice by telemetryusing an intravascular catheter PA-C 10 (DSI, MN), in which measurementwill be performed every three hours starting from 2 days before pumpplacement until 2 days after Ang pump placement. After this time, a newpump loaded with Ang II+peptide conjugate/TBM (with or withoutaromatic-cationic peptides)/aromatic-cationic peptide will be inserted,followed by another 2 days of recording to see if the peptide conjugate,TBM (with or without aromatic-cationic peptides) or aromatic-cationicpeptide has an effect on blood pressure.

Quantitative Pathology.

Ventricular tissues will be cut into transverse slices, and subsequentlyembedded with paraffin, sectioned, and subjected to Masson Trichromestaining. Quantitative analysis of fibrosis will be performed bymeasuring the percentage of blue-staining fibrotic tissue relative tothe total cross-sectional area of the ventricles.

Measurement of Mitochondrial Protein Carbonyl Groups.

For mitochondrial protein extraction, ventricular tissues will behomogenized in mitochondrial isolation buffer (1 mM EGTA, 10 mM HEPES,250 mM sucrose, 10 mM Tris-HCl, pH 7.4). The lysates will be centrifugedfor 7 minutes at 800 g in 4° C. The supernatants will be thencentrifuged for 30 minutes at 4000 g in 4° C. The crude mitochondriapellets will be resuspended in small volume of mitochondrial isolationbuffer, sonicated on ice to disrupt the membrane, and treated with 1%streptomycin sulfate to precipitate mitochondrial nucleic acids. TheOXISELECT™ Protein Carbonyl ELISA Kit (Cell Biolabs) will be used toanalyze 1 μg of protein sample per assay. The ELISA will be performedaccording to the instruction manual, with slight modification. Briefly,protein samples will be reacted with dinitrophenylhydrazine (DNPH) andprobed with anti-DNPH antibody, followed by HRP conjugated secondaryantibody. The anti-DNPH antibody and HRP conjugated secondary antibodyconcentrations will be 1:2500 and 1:4000, respectively.

Quantitative PCR.

Gene expression will be quantified by quantitative real-time PCR usingan Applied Biosystems 7900 thermocycler with Taqman Gene ExpressionAssays on Demand, which includes: PGCl-α (Mm00731216), TFAM(Mm004474X5), NRF-1 (Mm00447996), NRF-2 (Mm00487471), Collagen 1a2(Mm00483937), and ANP (Mm01255747). Expression assays will be normalizedto 18S RNA.

NADPH Oxidase Activity.

The NADPH oxidase assay will be performed as described elsewhere. Inbrief, 10 μg of ventricular protein extract will be incubated withdihydroethidium (DHE, 10 μM), sperm DNA (1.25 μg/mL), and NADPH (50 μM)in PBS/DTPA (containing 100 μM DTPA). The assay will be incubated at 37°C. in the dark for 30 minutes and the fluorescence will be detectedusing excitation/emission of 490/580 nm.

Western Immunoblots.

Cardiac protein extracts will be prepared by homogenization in lysisbuffer containing protease and phosphatase inhibitors on ice (1.5 mMKCl, 50 mM Tris HCl, 0.125% Sodium deoxycholate, 0.375% Triton X 100,0.15% NP40, 3 mM EDTA). The samples will be sonicated and centrifuged at10,000×g for 15 minutes at 4° C. The supernatant will be collected andthe protein concentration determined using a BCA assay (Pierce ThermoScientific, Rockford, Ill., U.S.A.). Total protein (25 μg) will beseparated on NuPAGE 4-12% Bis-Tris gel (Invitrogen) and transferred to0.45 μm PVDF membrane (Millipore), and then blocked in 5% non-fat drymilk in Tris-buffer solution with 0.1% Tween-20 for 1 hour. Primaryantibodies will be incubated overnight, and secondary antibodies will beincubated for 1 hour. The primary antibodies include: rabbit monoclonalanti-cleaved caspase-3 (Cell Signaling), mouse monoclonal anti-GAPDH(Millipore), rabbit polyclonal phospho-p3X MAP kinase (Cell Signaling),and mouse monoclonal anti-p38 (Santa Cruz Biotechnology). The enhancedchemiluminescence method (Thermo Scientific) will be used for detection.Image Quant ver.2.0 will be used to quantified the relative band densityas a ratio to GAPDH (internal control). All samples will be normalizedto the same cardiac protein sample.

It is anticipated that Ang-II will increase mitochondrial ROS inneonatal cardiomyocytes, which will be alleviated by treatment withpeptide conjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides). It is predicted that flow cytometryanalysis will demonstrate that Angiotensin II increased Mitosoxfluorescence (an indicator of mitochondrial superoxide) in neonatalcardiomyocytes. It is predicted that treatment with N-acetyl cysteine(NAC), a non-targeted antioxidant drug, will not show any effect on thelevel of mitochondrial ROS after Ang II. In contrast, it is anticipatedthat peptide conjugates, aromatic-cationic peptides or TBMs (with orwithout aromatic-cationic peptides) will reduce Ang II-inducedfluorescence to the level similar to that of saline-treated controlcardiomyocytes. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. These anticipated results will indicate that Ang IIinduced mitochondrial oxidative stress in cardiomyocytes can bealleviated by a mitochondrial targeted antioxidant.

Treatment with peptide conjugates, aromatic-cationic peptides or TBMs(with or without aromatic-cationic peptides) is anticipated toameliorate Ang II-induced cardiomyopathy despite the absence of bloodpressure lowering effect. To recapitulate hypertensive cardiomyopathy, apressor dose of Ang II (1.1 mg/kg/d) will be administered for 4 weeksvia subcutaneous continuous delivery with Alzet 1004 osmotic minipumps.It is predicted that intravascular telemetry will reveal that this doseof Ang II will significantly increase systolic and diastolic bloodpressure by 25-28 mm Hg above baseline. It is predicted that thesimultaneous administration of peptide conjugates (3 mg/kg/d),aromatic-cationic peptides (an equivalent molar dose ofaromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the peptide conjugate group)TBMs (an equivalent molar dose of TBM based on concentration of the TBMadministered in the peptide conjugate group) or TBMs in combination witharomatic-cationic peptides (e.g., an equivalent molar dose of TBM basedon the concentration of TBM administered in the peptide conjugatetreatment group and an equivalent molar dose of aromatic-cationicpeptide based on the concentration of aromatic-cationic peptideadministered in the peptide conjugate treatment group) will not have anyeffect on blood pressure.

The cardiac pathology will be examined by Masson trichrome staining,which demonstrated perivascular fibrosis and interstitial fibrosis after4 weeks of Ang II. It is anticipated that quantitative image analysis ofventricular fibrosis (blue staining on trichrome) will show that Ang IIsignificantly increases ventricular fibrosis, which is anticipated to befully attenuated by peptide conjugates, aromatic-cationic peptides orTBMs (with or without aromatic-cationic peptides). It is anticipatedthat administration of peptide conjugates of the present technology willhave synergistic effects in this regard compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone. The increase in cardiacfibrosis will be confirmed by quantitative PCR of the procollagen 1a2gene, the main component of fibrosis.

Consistent with the expectation that Ang II will induce mitochondrialROS in cardiomyocytes, it is predicted that chronic administration ofAng II for 4 weeks will significantly increase ventricular mitochondrialprotein carbonyl content, which is an indicator of protein oxidativedamage. It is anticipated that mitochondrial targeted antioxidantpeptide conjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides) will significantly reduce cardiacmitochondrial protein carbonyls. It is anticipated that administrationof peptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that TBMs (with or without aromatic-cationic peptides)or peptide conjugates act downstream of NADPH oxidase and will reduceactivation of p38 MAPK and apoptosis in response to Ang II. It isanticipated that consistent with previous reports, 4 weeks of Ang IIwill significantly increase cardiac NADPH oxidase activity, however, itis predicted this will not be changed by administration of peptideconjugates or TBMs (with or without aromatic-cationic peptides), whichsuggests that TBMs or peptide conjugate protection acts downstream ofNADPH oxidase.

Ang II has been shown to activate several mitogen activated proteinkinase (MAPK), such as p38. It is anticipated that administration of AngII for 4 weeks will increase phosphorylation of p38 MAPK, and thisphosphorylation will be significantly and nearly fully attenuated bypeptide conjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides), which suggests that MAP kinase is activatedthrough mitochondrial —ROS sensitive mechanisms. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

Mitochondrial ROS, either directly, or indirectly by activatingapoptosis signal regulating kinase, may induce apoptosis. It isanticipated that Ang II will induce cardiac apoptosis, which will beshown through an increase in cleaved caspase-3. It is also anticipatedthat peptide conjugates, aromatic-cationic peptides or TBMs (with orwithout aromatic-cationic peptides) will completely prevent theactivation of caspase-3 caused by Ang II. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that peptide conjugates, aromatic-cationic peptides orTBMs (with or without aromatic-cationic peptides) will partially rescueGαq overexpression-induced heart failure. Gαq protein is coupled toreceptors for catecholamines and Ang II, all of which are known to bekey mediators in hypertensive cardiovascular diseases. To extend theseobservations to a model of chronic catecholamine/Ang II stimulation, agenetic mouse model with cardiac specific overexpression of Gαq will beused, which causes heart failure in mice by 14-16 weeks of age. The Gαqmice in this study will have impairment of systolic function at 16 weeksage, which will be shown by a substantial decline in FS, withenlargement of the LV chamber, impairment of diastolic functionindicated by decreased Ea/Aa, and worsening of myocardial performanceindex (MPI). Peptide conjugates (3 mg/kg/d), aromatic-cationic peptides(an equivalent molar dose of aromatic-cationic peptide based onconcentration of the aromatic-cationic peptide administered in thepeptide conjugate group), TBMs (an equivalent molar dose of TBM based onconcentration of the TBM administered in the peptide conjugate group),or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) will be administered from 12 to 16 weeks of age, and itis predicted that these compounds will significantly ameliorate systolicfunction and improve myocardial performance. LV chamber enlargement isanticipated to be slightly reduced from treatment with peptideconjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides). It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects with respect topreventing or treating hypertensive cardiomyopathy or heart failure inmammalian subjects compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBMs in combination with aromatic-cationic peptides will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for preventing or treatingcardiomyopathy or heart failure in mammalian subjects.

Example 58: Compositions of the Present Technology Protect AgainstVessel Occlusion Injuries

This Example will demonstrate that the administration of TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates at the time ofrevascularization limits the size of the infarct during acute myocardialinfarction.

Men and women, 18 years of age or older, who present after the onset ofchest pain, and for whom the clinical decision is made to treat with arevascularization procedure (e.g., PCI or thrombolytics) will beeligible for enrollment. Patients may be STEMI (ST-Elevation MyocardialInfarction) or Non-STEMI. A STEMI patient will present with symptomssuggestive or a cutting off of the blood supply to the myocardium andalso if the patient's ECG shows the typical heart attack pattern of STelevation. The diagnosis is made therefore purely on the basis ofsymptoms, clinical examination and ECG changes. In the case of a Non-STelevation heart attack, the symptoms of chest pain can be identical tothat of a STEMI but the important difference is that the patient's ECGdoes not show the typical ST elevation changes traditionally associatedwith a heart attack. The patient often has a history of havingexperienced angina, but the ECG at the time of the suspected attack mayshow no abnormality at all. The diagnosis will be suspected on thehistory and symptoms and will be confirmed by a blood test which shows arise in the concentration of substances called cardiac enzymes in theblood.

Left ventricular and coronary angiography will be performed with the useof standard techniques, just before revascularization. Revascularizationwill be performed by PCI with the use of direct stenting. Alternativerevascularization procedures include, but are not limited to, balloonangioplasty; percutaneous transluminal coronary angioplasty; anddirectional coronary atherectomy.

After coronary angiography is performed but before the stent isimplanted, patients who meet the enrollment criteria are randomlyassigned to either the control group or the experimental group.Randomization is performed with the use of a computer-generatedrandomization sequence. Less than 10 minutes before direct stenting, thepatients in the experimental group receive an intravenous bolusinjection of the peptide conjugate, aromatic-cationic peptide or TBMs(with or without aromatic-cationic peptides). Patients will be equallyrandomized into any of the following treatment arms (for example, 0,0.001, 0.005, 0.01, 0.025, 0.05, 0.10, 0.25, 0.5, and 1.0 mg/kg/hour forpeptide conjugates and equivalent molar doses of aromatic-cationicpeptide, TBM, or TBM+aromatic-cationic peptides based on concentrationof the aromatic-cationic peptide and/or TBM administered in the peptideconjugate group). The compound will be administered as an IV infusionfrom about 10 minutes prior to reperfusion to about 3 hours post-PCL.Following the reperfusion period, the subject may be administered thecompound chronically by any means of administration, e.g., subcutaneousor IV injection.

The primary end point is the size of the infarct as assessed bymeasurements of cardiac biomarkers. Blood samples will be obtained atadmission and repeatedly over the next 3 days. Coronary biomarkers willbe measured in each patient. For example, the area under the curve (AUC)(expressed in arbitrary units) for creatine kinase and troponin Irelease (Beckman kit) may be measured in each patient by computerizedplanimetry. The principal secondary end point is the size of the infarctas measured by the area of delayed hyperenhancement that is seen oncardiac magnetic resonance imaging (MRI), assessed on day 5 afterinfarction. For the late-enhancement analysis, 0.2 mmol ofgadolinium-tetrazacyclododecanetetraacetic acid (Gd.DOTA) per kilogramwill be injected at a rate of 4 mL per second and will be flushed with15 mL of saline. Delayed hyperenhancement is evaluated 10 minutes afterthe injection of gadolinium Gd.DOTA with the use of a three dimensionalinversion-recovery gradient-echo sequence. The images are analyzed inshort axis slices covering the entire left ventricle.

Myocardial infarction will be identified by delayed hyperenhancementwithin the myocardium, defined quantitatively by an intensity of themyocardial postcontrast signal that is more than 2 SD above that in areference region of remote, non-infarcted myocardium within the sameslice. For all slices, the absolute mass of the infracted area will becalculated according to the following formula: infarct mass (in grams oftissue)=(hyperenhanced area [in square centimeters]) x slice thickness(in centimeters) x myocardial specific density (1.05 g per cubiccentimeter).

It is predicted that administration of peptide conjugates,aromatic-cationic peptides or TBMs (with or without aromatic-cationicpeptides) at the time of reperfusion will be associated with a smallerinfarct by some measures than that seen with placebo. It is anticipatedthat administration of peptide conjugates of the present technology willhave synergistic effects in this regard compared to that observed witheither aromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone. These results will show thatthe TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology, or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful for limiting infarct size during acute myocardial infarction.

Example 59: Compositions of the Present Technology Protect Against AcuteMyocardial Infarction Injury in a Rabbit Model

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates in protecting againstan acute myocardial infarction injury in a rabbit model.

New Zealand white rabbits will be used in this study. The rabbits willbe males and >10 weeks in age. Environmental controls in the animalrooms will be set to maintain temperatures of 61° to 72° F. and relativehumidity between 30% and 70%. Room temperature and humidity will berecorded hourly, and monitored daily. There will be approximately 10-15air exchanges per hour in the animal rooms. Photoperiod will be 12-hrlight/12-hr dark (via fluorescent lighting) with exceptions as necessaryto accommodate dosing and data collection. Routine daily observationswill be performed. Harlan Teklad, Certified Diet (2030C), rabbit dietwill be provided approximately 180 grams per day from arrival to thefacility. In addition, fresh fruits and vegetables will be given to therabbit 3 times a week.

Peptide conjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides) will be used as the test article. Dosingsolutions will be formulated and will be delivered via continuousinfusion (IV) at a constant rate (e.g., 50 μL/kg/min for peptideconjugates and equivalent molar doses of aromatic-cationic peptide, TBMor TBM+aromatic-cationic peptide based on concentration of thearomatic-cationic peptide and/or TBM administered in the peptideconjugate group). Normal saline (0.9% NaCl) will be used as a control.

The test/vehicle articles will be given intravenously, under generalanesthesia, in order to mimic the expected route of administration inthe clinical setting of AMI and PTCA. Intravenous infusion will beadministered via a peripheral vein using a Kd Scientific infusion pump(Holliston, Mass. 01746) at a constant volume (e.g., 50 μL/kg/min forpeptide conjugates and equivalent molar doses of aromatic-cationicpeptide, TBM or TBM+aromatic-cationic peptide based on concentration ofthe aromatic-cationic peptide and/or TBM administered in the peptideconjugate group).

The study followed a predetermined placebo and sham controlled design.In short, 10-20 healthy, acclimatized, male rabbits will be assigned toone of these study arms (approximately 2-10 animals per group). Arm A(n=4, CTRL/PLAC) includes animals treated with vehicle (vehicle; VEH,IV); Arm B (n=7, treated) includes animals treated with the compound(peptide conjugates, aromatic-cationic peptides or TBMs (with or withoutaromatic-cationic peptides)); Arm C (n=2, SHAM) includes sham operatedtime-controls treated with vehicle (vehicle; VEH, IV) or compound.

In all cases, treatments will be started approximately 30 minutes afterthe onset of a 30-minute ischemic insult (coronary occlusion) andcontinued for up to 3 hours following reperfusion. In all cases,cardiovascular function will be monitored both prior to and duringischemia, as well as for up to 180 minutes (3 hours) post-reperfusion.The experiments will be terminated 3 hours post-reperfusion (end ofstudy); irreversible myocardial injury (infarct size byhistomorphometery) at this time-point will be evaluated, and will be theprimary-end-point of the study.

It is anticipated that administration of peptide conjugates,aromatic-cationic peptides or TBMs (with or without aromatic-cationicpeptides) will result in decreased infarct size compared to the vehiclecontrol group. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBMs in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. These results will show that TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology, or pharmaceutically acceptable salts thereof, such asacetate, tartrate, or trifluoroacetate salts, are useful in methods forpreventing and treating acute myocardial infarction injury in mammaliansubjects.

Example 60: Compositions of the Present Technology and Cyclosporine inthe Treatment of Acute Myocardial Infarction Injury

This Example will demonstrate that the administration of a TBM (with orwithout aromatic-cationic peptides) or peptide conjugate, or apharmaceutically acceptable salt thereof such as acetate, tartrate, ortrifluoroacetate salt, and cyclosporine at the time of revascularizationlimits the size of the infarct during acute myocardial infarction.

Study Group.

Men and women, 18 years of age or older, who present within 6 hoursafter the onset of chest pain, who have ST-segment elevation of morethan 0.1 mV in two contiguous leads, and for whom the clinical decisionis made to treat with percutaneous coronary intervention (PCI) will beeligible for enrollment. Patients are eligible for the study whetherthey are undergoing primary PCI or rescue PCI. Occlusion of the affectedcoronary artery (Thrombolysis in Myocardial Infarction (TIMI) flow grade0) at the time of admission is also a criterion for inclusion.

Angiography and Revascularization.

Left ventricular and coronary angiography will be performed with the useof standard techniques, just before revascularization. Revascularizationwill be performed by PCI with the use of direct stenting. Alternativerevascularization procedures include, but are not limited to, balloonangioplasty; insertion of a bypass graft; percutaneous transluminalcoronary angioplasty; and directional coronary atherectomy.

Experimental Protocol.

After coronary angiography is performed but before the stent isimplanted, patients who meet the enrollment criteria are randomlyassigned to either the control group or the experimental group.Randomization will be performed with the use of a computer-generatedrandomization sequence. Less than 10 minutes before direct stenting, thepatients in the experimental group will receive an intravenous bolusinjection of the peptide conjugate/aromatic-cationic peptide/TBM (withor without aromatic-cationic peptides) and cyclosporine. The peptideconjugate, aromatic-cationic peptide or TBM (with or withoutaromatic-cationic peptides) will be dissolved in normal saline (finalconcentration, 25 mg/mL for peptide conjugate and equivalent molar dosesof aromatic-cationic peptide, TBM, or TBM and aromatic-cationic peptidebased on concentration of the aromatic-cationic peptide and/or TBMadministered in the peptide conjugate group) and will be injectedthrough a catheter that is positioned within an antecubital vein. Eitherseparately or simultaneously, cyclosporine (final concentration, 25 mgper milliliter will be injected through the catheter. Normal saline(0.9% NaCl) will be used as a control. The patients in the control groupreceive an equivalent volume of normal saline.

Infarct Size.

The primary end point will be the size of the infarct as assessed bymeasurements of cardiac biomarkers. Blood samples are obtained atadmission and repeatedly over the next 3 days. The area under the curve(AUC) (expressed in arbitrary units) for creatine kinase and troponin Irelease (Beckman kit) will be measured in each patient by computerizedplanimetry. The principal secondary end point will be the size of theinfarct as measured by the area of delayed hyperenhancement that is seenon cardiac magnetic resonance imaging (MRI), assessed on day 5 afterinfarction. For the late-enhancement analysis, 0.2 mmol ofgadolinium-tetrazacyclododecanetetraacetic acid (Gd.DOTA) per kilogramis injected at a rate of 4 mL per second and will be flushed with 15 mLof saline. Delayed hyperenhancement will be evaluated 10 minutes afterthe injection of Gd.DOTA with the use of a three dimensionalinversion-recovery gradient-echo sequence. The images are analyzed inshort axis slices covering the entire left ventricle.

Myocardial infarction will be identified by delayed hyperenhancementwithin the myocardium, defined quantitatively by an intensity of themyocardial postcontrast signal that is more than 2 SD above that in areference region of remote, non-infarcted myocardium within the sameslice. For all slices, the absolute mass of the infracted area will becalculated according to the following formula: infarct mass (in grams oftissue)=E (hyperenhanced area [in square centimeters]) x slice thickness(in centimeters) x myocardial specific density (1.05 g per cubiccentimeter).

Other End Points.

The whole-blood concentration of the TBM (with or withoutaromatic-cationic peptides) or peptide conjugate is measured immediatelyprior to PCI as well as at 1, 2, 4, 8 and 12 hours post PCI. Bloodpressure and serum concentrations of creatinine and potassium will bemeasured on admission and 24, 48, and 72 hours after PCI. Serumconcentrations of bilirubin, glutamyltransferase, and alkalinephosphatase, as well as white-cell counts, will be measured on admissionand 24 hours after PCI.

The cumulative incidence of major adverse events that occur within thefirst 48 hours after reperfusion are recorded, including death, heartfailure, acute myocardial infarction, stroke, recurrent ischemia, theneed for repeat revascularization, renal or hepatic insufficiency,vascular complications, and bleeding. The infarct-related adverse eventswill be assessed, including heart failure and ventricular fibrillation.In addition, 3 months after acute myocardial infarction, cardiac eventsare recorded, and global left ventricular function will be assessed byechocardiography (Vivid 7 systems; GE Vingmed).

It is predicted that administration of the peptide conjugate,aromatic-cationic peptide, or TBMs (with or without aromatic-cationicpeptides) along with cyclosporine at the time of reperfusion will beassociated with a smaller infarct by some measures than that seen withplacebo. It is anticipated that administration of peptide conjugates ofthe present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone. These results will show that TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology, or pharmaceutically acceptable salts thereof, such asacetate, tartrate, or trifluoroacetate salts, are useful in combinationwith cyclosporine useful in methods for the treatment of myocardialinfarction.

Example 61: Compositions of the Present Technology and Cyclosporine inthe Treatment of Nephrotoxicity in Transplant Patients

This Example will demonstrate the use of cyclosporine and (i) TBMs (withor without aromatic-cationic peptides) or (ii) peptide conjugates alongto treat nephrotoxicity in transplant patients.

To prevent organ or tissue rejection after transplant, patients oftenreceive a regimen of the immunosuppressive drug cyclosporine.Cyclosporine levels are established and maintained in the subject atlevels to effectively suppress the immune system. However,nephrotoxicity is a concern for these subjects, and the level of thedrug in the subject's blood is monitored carefully. Cyclosporine dosesare adjusted accordingly in order to not only prevent rejection, butalso to deter these potentially damaging side effects. Typically, anadult transplant patient receives cyclosporine as follows: IV: 2 to 4mg/kg/day IV infusion once daily over 4 to 6 hours, or 1 to 2 mg/kg IVinfusion twice a day over 4 to 6 hours, or 2 to 4 mg/kg/day as acontinuous IV infusion over 24 hours. Capsules: 8 to 12 mg/kg/day orallyin 2 divided doses. Solution: 8 to 12 mg/kg orally once daily. In somepatients, doses can be titrated downward with time to maintenance dosesas low as 3 to 5 mg/kg/day. In some patients, the tolerance forcyclosporine is poor, and cyclosporine therapy must be discontinued, thedosage lowered, or the dosage regimen cycled so as to preventdestruction of the subject's kidney.

This Example demonstrates the effects of a TBM (with or withoutaromatic-cationic peptides) or peptide conjugate, or a pharmaceuticallyacceptable salt thereof, such as acetate, tartrate, or trifluoroacetatesalt, together with cyclosporine on post-transplant organ health (e.g.,ischemia-reperfusion injury post transplant and organ rejection), aswell as kidney health (e.g., nephrotoxic effects of cyclosporine). It isanticipated that administering a TBM (with or without aromatic-cationicpeptides) or peptide conjugate will have a protective effect on thetransplant organ or tissue, and on kidney health during cyclosporinetreatment.

Transplant subjects receiving cyclosporine pursuant to standard pre- andpost-transplant procedures will be divided into groups. Atherapeutically effective amount of a peptide conjugate orpharmaceutically acceptable salt thereof such as acetate, tartrate, ortrifluoroacetate salt; aromatic-cationic peptide; or TBMs (with orwithout aromatic-cationic peptides) will be administered to subjectsprior to, during and/or after transplant. Subjects will be monitored forhealth and function of the transplanted tissue or organ, as well as theincidence and severity of nephrotoxicity often seen with prolongedcyclosporine administration.

It is predicted that subjects who receive the peptide conjugate,aromatic-cationic peptide or TBMs (with or without aromatic-cationicpeptides) will have a healthier transplanted organ or tissue, and/orwill be able to maintain a higher and/or more consistent cyclosporinedosage for longer periods of time compared to subjects who do notreceive the compounds. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. These results will show that TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology, or pharmaceutically acceptable salts thereof, such asacetate, tartrate, or trifluoroacetate salts, are useful in combinationwith cyclosporine is useful in methods for treating nephrotoxicity intransplant patients.

Example 62: Compositions of the Present Technology Facilitate ElectronTransfer

ATP synthesis in the electron transport chain (ETC) is driven byelectron flow through the protein complexes of the ETC which can bedescribed as a series of oxidation/reduction processes. Rapid shuntingof electrons through the ETC is important for preventingshort-circuiting that would lead to electron escape and generation offree radical intermediates. The rate of electron transfer (ET) betweenan electron donor and electron acceptor decreases exponentially with thedistance between them, and superexchange ET is limited to 20 angstrom.Long-range ET can be achieved in a multi-step electron hopping process,where the overall distance between donor and acceptor is split into aseries of shorter, and therefore faster, ET steps. In the ETC, efficientET over long distances is assisted by cofactors that are strategicallylocalized along the inner mitochondrial membrane, including FMN, FeSclusters, and hemes. Aromatic amino acids such as Phe, Tyr and Trp canalso facilitate electron transfer to heme through overlapping π clouds,and this was specifically shown for cytochrome c. Amino acids withsuitable oxidation potential (Tyr, Trp, Cys, Met) can act as steppingstones by serving as intermediate electron carriers. In addition, thehydroxyl group of Tyr can lose a proton when it conveys an electron, andthe presence of a basic group nearby, such as Lys, can result inproton-coupled ET which is even more efficient.

It is hypothesized that the distribution of TBMs or peptide conjugatesamong the protein complexes in the IMM allows it to serve as additionalan relay station to facilitate ET. This will be demonstrated using thekinetics of cytochrome c reduction (monitored by absorbancespectroscopy) as a model system, with the TBMs or peptide conjugatefacilitating ET. Addition of N-acetylcysteine (NAC) as a reducing agentis anticipated to result in time-dependent increase in absorbance at 550nm. It is further anticipated that the addition of the TBM (with orwithout aromatic-cationic peptides) or peptide conjugate alone at 100 μMconcentrations will not reduce cytochrome c, but will dose-dependentlyincrease the rate of NAC-induced cytochrome c reduction, suggesting thatthe compound does not donate an electron but increases the speed ofelectron transfer.

This Example will further demonstrate the effect of TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates on therestoration of mitochondrial respiration and ATP synthesis followingischemia-reperfusion (IR) injury in rats. Animals will be subjected tobilateral occlusion of renal artery for 45 minutes followed by 20minutes or 1 hour of reperfusion. Subjects will receive saline vehicle,a peptide conjugate (2.0 mg/kg s.c.), an aromatic-cationic peptide (anequivalent molar dose of aromatic-cationic peptide based onconcentration of the aromatic-cationic peptide administered in thepeptide conjugate group) TBM (an equivalent molar dose of TBM based onconcentration of the TBM administered in the peptide conjugate group),or TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the peptide conjugate treatment group and an equivalentmolar dose of aromatic-cationic peptide based on the concentration ofaromatic-cationic peptide administered in the peptide conjugatetreatment group) 30 minutes before ischemia and again at the time ofreperfusion (n=4-5 in each group). It is anticipated that the peptideconjugate, aromatic-cationic peptide or TBMs (with or withoutaromatic-cationic peptides) will improve oxygen consumption and ATPsynthesis. It is anticipated that administration of peptide conjugatesof the present technology will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMs(alone or in combination with aromatic-cationic peptides). It isanticipated that administration of TBM in combination witharomatic-cationic peptides will have synergistic effects in this regardcompared to that observed with either aromatic-cationic peptides or TBMsalone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods comprising electronscavenging electron transfer.

Example 63: Compositions of the Present Technology Enhance MitochondrialReduction Potential

The redox environment of a cell depends on its reduction potential andreducing capacity. Redox potential is highly compartmentalized withinthe cell, and the redox couples in the mitochondrial compartment aremore reduced than in the other cell compartments and are moresusceptible to oxidation. Glutathione (GSH) is present in mMconcentrations in mitochondria and is considered the major redox couple.The reduced thiol group —SH can reduce disulfide S—S groups in proteinsand restore function. The redox potential of the GSH/GSSG couple isdependent upon two factors: the amounts of GSH and GSSG, and the ratiobetween GSH and GSSG. As GSH is compartmentalized in the cell and theratio of GSH/GSSG is regulated independently in each compartment,mitochondrial GSH (mGSH) is the primary defense against mitochondrialoxidative stress. Mitochondrial GSH redox potential becomes moreoxidizing with aging, and this is primarily due to increase in GSSGcontent and decrease in GSH content.

It is anticipated that TBMs (with or without aromatic-cationic peptides)and peptide conjugates of the present technology will enhancemitochondrial reduction potential in vitro in isolated mitochondrial andin vivo in cultured cells and animal subjects. These results will showthat TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology, or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods for enhancing mitochondrial reduction potential.

Example 64: Compositions of the Present Technology Reduce MV-InducedMitochondrial Oxidation

This Example will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology reducemechanical ventilation (MV)-induced mitochondrial oxidation.

Experimental Design:

Murine subjects will be treated as follows:

-   -   1. Normal, mobile mice: Normal, mobile mice will be randomly        divided into four groups, A-E, with 8 mice per group. Group A        mice will receive an injection of saline vehicle; Group B mice        will receive an i.p. injection of the peptide conjugate; Group C        mice will receive an i.p. injection of aromatic-cationic        peptide; Group D mice will receive an i.p. injection of TBM;        Group E mice will receive an i.p. injection of        TBM+aromatic-cationic peptide.    -   2. Hind limb casted mice: Mouse hind limbs will be immobilized        by casting for 14 days, thereby inducing hind limb muscle        atrophy. Casted mice will receive an i.p. injection of saline        vehicle (0.3 mL), peptide conjugate, aromatic-cationic peptide,        TBM, or TBM with aromatic-cationic peptides. A control group of        untreated mice will be also used in this experiment.

To demonstrate that mitochondrial ROS production plays a role inimmobilization-induced skeletal muscle atrophy, mice will be randomlyassigned to one of three experimental groups (n=24/group): 1) notreatment (control) group; 2) 14 days of hind limb immobilization group(cast); and 3) 14 days of hind-limb immobilization group treated withthe mitochondrial-targeted antioxidant peptide conjugate,aromatic-cationic peptide, TBM, or TBM+aromatic-cationic peptides(CasHSS). Subjects will receive s.c. injections of saline vehicle (0.3mL) or the peptide conjugate, aromatic-cationic peptide or TBM (alone orin combination with aromatic-cationic peptides) (1.5 mg/kg for thepeptide conjugate and equivalent molar doses of aromatic-cationicpeptide and/or TBM based on concentration of the aromatic-cationicpeptide or TBM administered in the peptide conjugate group) administeredonce daily during the immobilization period.

Immobilization.

Mice will be anesthetized with gaseous isoflurane (3% induction,0.5-2.5%) maintenance). Anesthetized animals will be cast-immobilizedbilaterally with the ankle joint in the plantar-flexed position toinduce maximal atrophy of the soleus and plantaris muscle. Both hindlimbs and the caudal fourth of the body will be encompassed by a plastercast. A thin layer of padding will be placed underneath the cast inorder to prevent abrasions. In addition, to prevent the animals fromchewing on the cast, one strip of fiberglass material will be appliedover the plaster. The mice will be monitored on a daily basis for chewedplaster, abrasions, venous occlusion, and problems with ambulation.

Preparation of Permeabilized Muscle Fibers.

Permeabilized muscle fibers will be prepared as previously described.Korshunov, et al., FEBS Lett 416:15-18, 1997; Tonkonogi, et al.,Pfliigers Arch 446:261-269, 2003. Briefly, the muscle will be trimmed ofconnective tissue and cut down to fiber bundles (4-8 mg wet wt). Under amicroscope and using a pair of extra-sharp forceps, the muscle fiberswill be gently separated in ice-cold buffer X containing 60 mM K-MES, 35mM KCl, 7.23 mM K₂EGTA, 2.77 mM CaK₂EGTA, 20 mM imidazole, 0.5 mM DTT,20 mM taurine, 5.7 mM ATP, 15 mM PCr, and 6.56 mM MgCl₂.6H₂O (pH 7.1,295 mosmol/kg H₂O) to maximize surface area of the fiber bundle. Topermeabilize the myofibers, each fiber bundle will be incubated inice-cold buffer X containing 50 μg/mL saponin on a rotator for 30minutes at 4° C. The permeabilized bundles will be washed in ice-coldbuffer Z, containing 110 mM K-MES, 35 mM KCl, 1 mM EGTA, 5 mM K₂HPO₄,and 3 mM MgCl₂, 0.005 mM glutamate, and 0.02 mM malate and 0.5 mg/mLBSA, pH 7.1.

Mitochondrial Respiration in Permeabilized Fibers.

Respiration will be measured polarographically in a respiration chambermaintained at 37° C. (Hansatech Instruments, United Kingdom). After therespiration chamber will be calibrated, permeabilized fiber bundles willbe incubated with 1 mL of respiration buffer Z containing 20 mM creatineto saturate creatine kinase (Saks, et al., Mol. Cell Biochem.184:81-100, 1998; Walsh, et al., J. Physiol. 537:971-978, 2001). Fluxthrough complex I will be measured using 5 mM pyruvate and 2 mM malate.The maximal respiration (state 3), defined as the rate of respiration inthe presence of ADP, will be initiated by adding 0.25 mM ADP to therespiration chamber. Basal respiration (state 4) will be determined inthe presence of 10 μg/mL oligomycin to inhibit ATP synthesis. Therespiratory control ratio (RCR) will be calculated by dividing state 3by state 4 respiration.

Mitochondrial ROS Production.

Mitochondrial ROS production will be determined using AMPLEX™ Red(Molecular Probes, Eugene, Oreg., U.S.A.). The assay will be performedat 37° C. in 96-well plates using succinate as the substrate. Superoxidedismutase (SOD) will be added at 40 units/mL to convert all superoxideinto H₂O₂. Resorufin formation (AIVIPLEX™ Red oxidation by H₂O₂) will bemonitored at an excitation wavelength of 545 nm and an emissionwavelength of 590 nm using a multi-well plate reader fluorometer(SpectraMax, Molecular Devices, Sunnyvale, Calif., U.S.A.). The level ofResorufin formation will be recorded every 5 minutes for 15 minutes, andH₂O₂ production will be calculated with a standard curve.

It is anticipated that the peptide conjugate, aromatic-cationic peptideor TBMs (with or without aromatic-cationic peptides) will have no effecton normal skeletal muscle size or mitochondrial function, and that thepeptide conjugate, aromatic-cationic peptide or TBMs (with or withoutaromatic-cationic peptides) will prevent oxidative damage and associatedmuscle weakness induced by hind limb immobilization (e.g., atrophy,contractile dysfunction, etc.). It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that the peptide conjugate, aromatic-cationic peptideor TBMs (with or without aromatic-cationic peptides) will have no effecton normal soleus muscle weight, the respiratory coupling ratio (RCR),mitochondrial state 3 respiration, or mitochondrial state 4 respiration,in mobile mice. RCR is the respiratory quotient ratio of state 3 tostate 4 respiration, as measured by oxygen consumption. Likewise, it isanticipated that the peptide conjugate, aromatic-cationic peptide orTBMs (with or without aromatic-cationic peptides) will not causevariable effects on muscle fibers of different size in a normal soleusmuscle, or on plantaris muscle weight, the respiratory coupling ratio(RCR), mitochondrial state 3 respiration, or mitochondrial state 4respiration. Similarly, it is anticipated that the peptide conjugate,aromatic-cationic peptide or TBMs (with or without aromatic-cationicpeptides) will not have any variable effects to the muscle fibers ofdifferent size in normal plantaris muscle fiber tissue.

It is anticipated that hind limb casting for 7 days will cause asignificant decrease in soleus muscle weight and mitochondrial state 3respiration, both of which are anticipated to be reversed byadministration of the peptide conjugate, aromatic-cationic peptide orTBMs (with or without aromatic-cationic peptides). It is anticipatedthat casting for 7 days will significantly increase H₂O₂ production bymitochondria isolated from soleus muscle, which is anticipated to beprevented by the peptide conjugate, aromatic-cationic peptide or TBMs(with or without aromatic-cationic peptides). It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

Casting is also anticipated to significantly increase oxidative damagein soleus muscle, as measured by lipid peroxidation via 4-hydroxynonenal(4-HNE). It is anticipated that this effect will be overcome byadministration of the peptide conjugate, aromatic-cationic peptide orTBMs (with or without aromatic-cationic peptides). Moreover, it isanticipated that casting will significantly increase protease activityin the soleus muscle, promoting muscle degradation and atrophy, and thatthis effect will be attenuated or prevented by administration of thepeptide conjugate, aromatic-cationic peptide or TBMs (with or withoutaromatic-cationic peptides). It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

It is anticipated that calpain-1, caspase-3 and caspase-12 proteolyticdegradation of muscle, respectively, will be all prevented by treatmentwith the peptide conjugate, aromatic-cationic peptide or TBMs (with orwithout aromatic-cationic peptides). It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

These results will show that administering peptide conjugates,aromatic-cationic peptides or TBMs (with or without aromatic-cationicpeptides) to subjects with MV-induced or disuse-induced increases inmitochondrial ROS production reduces protease activity and attenuatesskeletal muscle atrophy and contractile dysfunction. The results willfurther show that treatment of animals with the mitochondrial-targetedantioxidant peptide conjugate, aromatic-cationic peptide or TBMs (withor without aromatic-cationic peptides) is useful in preventing theatrophy of type I, IIa, and IIx/b skeletal muscle fibers, and thatprevention of MV-induced and disuse-induced increases in mitochondrialROS production protects the diaphragm from MV-induced decreases indiaphragmatic specific force production at both sub-maximal and maximalstimulation frequencies. It is anticipated that administration ofpeptide conjugates of the present technology will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs (alone or in combination witharomatic-cationic peptides). It is anticipated that administration ofTBM in combination with aromatic-cationic peptides will have synergisticeffects in this regard compared to that observed with eitheraromatic-cationic peptides or TBMs alone.

As such, TBMs (with or without aromatic-cationic peptides) or peptideconjugates of the present technology, or pharmaceutically acceptablesalts thereof, such as acetate, tartrate, or trifluoroacetate salts, areuseful in methods for treating or preventing MV-induced anddisuse-induced mitochondrial ROS production in the diaphragm and otherskeletal muscles.

Example 65: Compositions of the Present Technology Reduce the AnatomicZone of No-Reflow Following Ischemia/Reperfusion in the Brain

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in protecting a subject from an anatomic zone of no-reflowcaused by ischemia-reperfusion in the brain.

Cerebral ischemia initiates a cascade of cellular and molecular eventsthat lead to brain damage. One such event is an anatomic zone ofno-reflow. Cerebral ischemia will be induced by occlusion of the rightmiddle cerebral artery for 30 minutes. Wild-type (WT) mice will be giveneither saline vehicle (Veh), peptide conjugate, aromatic-cationicpeptide, TBM, TBM and aromatic-cationic peptide (2-5 mg/kg for thepeptide conjugate and equivalent molar doses of aromatic-cationicpeptide and/or TBM based on concentration of the aromatic-cationicpeptide or TBM administered in the peptide conjugate group) i.p. at 0,6, 24 and 48 hours after ischemia. Mice will be sacrificed 3 days afterischemia, and the brains sliced transversely into 6-8 sections. Sectionswill be photographed under ultraviolet light to identify the region ofno-reflow. The areas of no-reflow in each slice will be digitized usingImage J (supplier Rasband WS, Image J, National Institutes of Health,http://rsb.info.nih.gov/ij/). The areas in each slice will be multipliedby the weight of the slice and the results will be summed in order toobtain the mass of the no-reflow areas.

It is predicted that treatment of wild type mice with the peptideconjugate, aromatic-cationic peptide or TBMs (with or withoutaromatic-cationic peptides) will result in a significant reduction ininfarct volume and prevent or reduce the anatomic zone of no-reflow. Itis anticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone. These results willshow that the TBMs (with or without aromatic-cationic peptides) orpeptide conjugates of the present technology, or pharmaceuticallyacceptable salts thereof, such as acetate, tartrate, or trifluoroacetatesalts, are useful in methods for reducing the incidence of no-reflowcaused by ischemia-reperfusion in the brain.

Example 66: Compositions of the Present Technology Reduce the AnatomicZone of No-Reflow Following Ischemia/Reperfusion in the Kidney

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in protecting a subject from an anatomic zone of no-reflowcaused by ischemia-reperfusion in the kidney.

Sprague Dawley rats (250-300 g) will be assigned to the followinggroups: (1) sham surgery group without I/R; (2) I/R+saline vehicletreatment; (3) FR+peptide conjugate treatment; (4) I/R+aromatic-cationicpeptide treatment; (5) I/R+TBM treatment; (6) I/R+TBM+aromatic-cationicpeptides. The peptide conjugate (3 mg/kg, dissolved in saline),aromatic-cationic peptide (an equivalent molar dose of aromatic-cationicpeptide based on the concentration of the aromatic-cationic peptideadministered in the peptide conjugate group), TBM (an equivalent molardose of TBM based on the concentration of the TBM administered in thepeptide conjugate group), or TBMs in combination with aromatic-cationicpeptides (e.g., an equivalent molar dose of TBM based on theconcentration of TBM administered in the peptide conjugate treatmentgroup and an equivalent molar dose of aromatic-cationic peptide based onthe concentration of aromatic-cationic peptide administered in thepeptide conjugate treatment group) will be administered to rats 30minutes before ischemia and immediately before onset of reperfusion. Thecontrol rats will be given saline vehicle on the same schedule. Ratswill be anesthetized with a mixture of ketamine (90 mg/kg, i.p.) andxylazine (4 mg/kg, i.p.). The left renal vascular pedicle will beoccluded temporarily using a micro-clamp for 30 or 45 min. At the end ofthe ischemic period, reperfusion will be established by removing of theclamp. At that time, the contralateral right kidney will be removed.After 24 hours reperfusion, animals will be sacrificed and blood sampleswill be obtained by cardiac puncture. Renal function will be determinedby blood urea nitrogen (BUN) and serum creatinine (BioAssay SystemsDIUR-500 and DICT-500).

Analysis of No-reflow Zones, and Necrosis. The kidneys will be slicedtransversely into 6-8 sections. Sections will be photographed underultraviolet light to identify the region of no-reflow. The areas ofno-reflow in each slice are digitized using Image J (supplier RasbandWS, Image J, National Institutes of Health,http://rsb.info.nih.gov/ij/). The areas in each slice will be multipliedby the weight of the slice and the results will be summed in order toobtain the mass of the no-reflow areas.

It is predicted that treatment with the peptide conjugate,aromatic-cationic peptide or TBMs (with or without aromatic-cationicpeptides) will prevent or reduce the anatomic zone of no-reflow in thekidney. It is further predicted that one or more of BUN, serumcreatinine, and glomerular filtration rate will improve in subjectstreated with the peptide conjugate, aromatic-cationic peptide or TBMs(with or without aromatic-cationic peptides) as compared to untreatedcontrol subjects. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone. As such, the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology, orpharmaceutically acceptable salts thereof, such as acetate, tartrate, ortrifluoroacetate salts, are useful in methods for reducing the incidenceof no-reflow caused by ischemia-reperfusion in the kidney.

Example 67: Compositions of the Present Technology Protect Against theNo Re-Flow Phenomenon in Humans

This Example will demonstrate the use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology at the time of revascularization of ischemic tissue to limitthe size of the anatomic zone of no-reflow in human subjects.

For treatment of acute myocardial infarction (AMI), the use ofmechanical recanalization of the affected artery restores epicardialcoronary blood flow to ischemic myocardium (TIMI Flow Grade 3) in morethan 90% of patients. However, these reperfusion methods do not addressthe important ancillary problem of restoration of blood flow downstreamat the level of the capillary bed. During or following primarypercutaneous coronary intervention (PCI), microcirculatory dysfunctionis observed in 20-40% of patients. The lack of ST-segment elevationresolution after angioplasty with stenting is a marker of microvascularproblems and is associated with a poor clinical prognosis. In STEMI,failure to achieve myocardial reperfusion despite the presence of apatent coronary artery has been called the “no-reflow” phenomenon.

Study Group.

Men and women, 18 years of age or older, who present within 6 hoursafter the onset of chest pain, who have ST-segment elevation of morethan 0.1 mV in two contiguous leads, and for whom the clinical decisionis made to treat with PCI will be eligible for enrollment. Patients willbe eligible for the study whether they are undergoing primary PCI orrescue PCI. Occlusion of the affected coronary artery (Thrombolysis inMyocardial Infarction [TIMI] flow grade 0) at the time of admission willalso be a criterion for inclusion.

Angiography and Revascularization.

Left ventricular and coronary angiography will be performed with the useof standard techniques, just before revascularization. Revascularizationwill be performed by PCI with the use of direct stenting. Alternativerevascularization procedures include, but are not limited to, balloonangioplasty; insertion of a bypass graft; percutaneous transluminalcoronary angioplasty; and directional coronary atherectomy

Experimental Protocol.

After coronary angiography is performed but before the stent isimplanted, patients who meet the enrollment criteria will be randomlyassigned to the control group; the peptide conjugate treatment group;the aromatic-cationic peptide group; the TBM treatment group;TBM+aromatic-cationic peptide treatment group. Randomization will beperformed with the use of a computer-generated randomization sequence.Less than 10 minutes before direct stenting, the patients in theexperimental group receive an intravenous bolus injection of the peptideconjugate, aromatic-cationic peptide or TBM (alone or in combinationwith aromatic-cationic peptide). The compound will be dissolved innormal saline (final concentration, 25 mg per milliliter for peptideconjugate and equivalent molar doses of aromatic-cationic peptide and/orTBM based on concentration of the aromatic-cationic peptide or TBMadministered in the peptide conjugate group) and will be injectedthrough a catheter that is positioned within an antecubital vein. Thepatients in the control group receive an equivalent volume of normalsaline.

No Re-Flow Zone.

The primary end point will be the size of the anatomic zone ofno-reflow. No re-flow will be assessed by one or more imagingtechniques. Re-flow phenomenon will be assessed using myocardialcontrast echocardiography, coronary angiography, myocardial blush,coronary doppler imaging, electrocardiography, nuclear imagingsingle-photon emission CT, using thallium or technetium-99m, or PET. A1.5-T body MRI scanner will be used to perform cardiac MRI in order toassess ventricular function, myocardial edema (area at risk),microvascular obstruction and infarct size. Post-contrast delayedenhancement will be used on day 4±1, day 30±3 and 6+1.5 months aftersuccessful PCI and stenting to quantify infracted myocardium. This willbe defined quantitatively by an intensity of the myocardialpost-contrast signal that is more than 2 SD above that in a referenceregion of remote, non-infarcted myocardium within the same slice.Standard extracellular gadolinium-based contrast agents will be used ata dose of 0.2 mmol/kg. The 2D inversion recovery prepared fast gradientecho sequences will be used at the following time points: (1) early(approximately 2 minutes after contrast injection) for evaluation ofmicrovascular obstruction. Single shot techniques may be considered ifavailable and (2) late (approximately 10 minutes after contrastinjection) for evaluation of infarct size.

It is predicted that administration of the peptide conjugate,aromatic-cationic peptide or TBMs (with or without aromatic-cationicpeptides) at the time of reperfusion will be associated with a smalleranatomic zone of no-reflow than that seen with placebo. It isanticipated that administration of peptide conjugates of the presenttechnology will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs (alone or incombination with aromatic-cationic peptides). It is anticipated thatadministration of TBM in combination with aromatic-cationic peptideswill have synergistic effects in this regard compared to that observedwith either aromatic-cationic peptides or TBMs alone. As such, the TBMs(with or without aromatic-cationic peptides) or peptide conjugates ofthe present technology, or pharmaceutically acceptable salts thereof,such as acetate, tartrate, or trifluoroacetate salts, are useful inmethods for reducing the incidence of no-reflow caused byischemia-reperfusion in the heart.

Example 68—Use of Compositions of the Present Technology in theTreatment of Drug-Induced Hyperalgesia in Humans

This Example will demonstrate use of TBMs (with or withoutaromatic-cationic peptides) or peptide conjugates of the presenttechnology in the treatment of hyperalgesia in human subjects.

Patients will be recruited to the study as they present in clinic withchronic (>6 months' duration), spontaneous, ongoing, vincristine-relatedpain. Those enrolled will rate their daily maximum level of pain at 4 orgreater on a visual analog scale (VAS). The patients will be screenedfor their willingness to enroll in the study, and informed consent willbe obtained. Healthy subjects will also be recruited for collection ofcomparison data. No subjects in either the patient or comparison groupwill have known risk factors for any other cause of peripheralneuropathy, including diabetes, AIDS, chronic alcoholism, or previousradiation exposure.

After a focused interview about the history of the patient's cancer andtreatment, the patient will be asked to describe sensory symptoms bychoosing from a list of ideal type word descriptors. Ongoing and dailymaximum pain intensity will be rated on a VAS with prompts of “no pain”at the bottom and “most imaginable” at the top. The areas of pain andsensory disturbances will be drawn by each patient on a standardizedbody map. Similar to previous observations in patients treated withpaclitaxel, subjects with vincristine-induced peripheral neuropathy arepredicted to identify the following three zones of sensation:

a) The painful area: The zone of ongoing pain located on the tips of thefingers and/or toes. The tip of the index finger is expected to beinvolved in all patients and will be used as the test site in this zone.

b) The border area: Adjacent and proximal to, but distinct from thepainful area, represented by nonpainful sensory disturbances and locatedin the palms and/or soles of the feet. The thenar eminence is expectedto be involved in all patients and will be used as the test site in thiszone.

c) The nonpainful area: Adjacent and proximal to, but distinct from theborder area, reported by the patient to feel “normal.” This site isexpected to be always proximal to the wrists and/or ankles. Sensorytesting will be conducted on the volar surface of the arm.

The tip of the index finger, thenar eminence, and volar forearm, will betested in normal subjects for comparison. Patients will be specificallyqueried about the stimuli that provoked pain or caused an exacerbationof ongoing pain in these regions, including the effects that clothing,bed linens, bathing, and normal activities of daily living cause. Eachzone will be examined for any physical changes, such as scaling, fingerclubbing, and erythema, which will be documented. The areas of sensorydisturbance will be physically probed by light touch with a camel hairbrush and by manual massage to screen for the presence of allodynia orhyperalgesia.

Touch and Sharpness Detection Thresholds—

Touch detection thresholds will be determined with von Freymonofilaments using the up/down method as previously reported. Startingwith a bending force of 0.02 g, each monofilament will be applied to aspot on the skin less than 2 mm in diameter for approximately onesecond. The force of the filament detected four consecutive times willbe assigned as the touch detection threshold. Sharpness detection willbe determined using weighted 30-gauge metal cylinders. Briefly, the tipof 30-gauge needles (200 mm diameter) will be filed to produce flat,cylindrical ends and the luers will be fitted to calibrated brassweights with the desired force (100, 200, and 400 mN) level for eachstimulus. Each loaded needle will be placed inside a separate 10 ccsyringe where it will be able to move freely. Each stimulus will beapplied for one second perpendicular to the skin 10 times within eacharea of interest in a pseudorandom order. The subjects will indicatewhether the stimulus is perceived as touch, pressure, sharp, or other.The percentages of each reply will be calculated and then combined intogroup grand means for comparison. The 50% sharpness detection thresholdwill be calculated as the weighted needle that caused five or more sharpresponses after 10 consecutive stimuli.

Grooved Pegboard Test—

Manual dexterity will be assessed with the grooved pegboard test.Subjects will be instructed to fill a five-by-five slotted pegboard inan ordered fashion and the times for both dominant and non-dominanthands will be recorded.

Thermal Detection Thresholds—

The threshold for heat pain will be determined using the Marstocktechnique. A radiometer will be used at the outset of testing toascertain the baseline skin temperature at all testing sites. All testsand measurements will be conducted at room temperature 22° C. Thermalramps will be applied using a 3.6×3.6 cm Peltier thermode from abaseline temperature of 32° C. Skin heating will be at a ramp of 0.30°C./s, and skin cooling will be at a ramp of −0.5° C./s. Subjects will beinstructed to signal when the stimulus is perceived as first becomingwarmer and then painfully hot, or as first becoming cooler and thenpainfully cold. If a subject fails to reach a given threshold before thecutoff temperature of 51.5° C. for the ascending ramp or 3° C. held for10 seconds in the cooling test, the cutoff values will be assigned forany that are not reached. The final threshold value for each skinsensation in each patient will be determined by averaging the results ofthree heating and cooling trials.

Statistical Analysis—

The thresholds for touch detection will be compared using nonparametricmethods (Wilcoxon's test). The sharpness detection, thermal thresholds,and times in the grooved pegboard tests will be compared using analysisof variance and post hoc comparison of the means with Duncan's multiplerange tests. Comparisons of mechanical and thermal thresholds will beperformed between healthy subjects and patients for the different areasof the tested skin. Further analyses will be performed between glabrousand volar skin within the patient group. For every comparison performedin the present study, p<0.05 will be considered significant.

Following initial assessment of the above criteria, subjects will bedivided into the following groups:

a) Healthy controls

b) No treatment

c) Vehicle-only placebo, administered s.c., once daily for 14 days

d) peptide conjugate, 10 mg/kg, administered s.c., once daily for 14days

e) aromatic-cationic peptide (equivalent molar doses ofaromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 10 mg/kg peptide conjugategroup), administered s.c., once daily for 14 days

f) TBM (equivalent molar doses of TBM based on concentration of the TBMadministered in the 10 mg/kg peptide conjugate group), administereds.c., once daily for 14 days

g) TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the 10 mg/kg peptide conjugate treatment group and anequivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 10 mg/kgpeptide conjugate treatment group), administered s.c., once daily for 14days.

Following the 14 day treatment period, subjects will be re-assessedaccording to the above criteria, with statistical analysis as describedabove.

Results—

It is expected that neuropathy subjects administered the peptideconjugate, aromatic-cationic peptide or TBM (with or withoutaromatic-cationic peptides) for a period of 14 days will report areduction in hyperalgesia symptoms compared to subjects administered notreatment or a vehicle-only placebo. The reduction in hyperalgesia willbe manifested in improved scoring for touch and sharpness detectionthresholds, grooved pegboard tests, and thermal detection tests comparedto control subjects. It is anticipated that administration of peptideconjugates of the present technology will have synergistic effects inthis regard compared to that observed with either aromatic-cationicpeptides or TBMs (alone or in combination with aromatic-cationicpeptides). It is anticipated that administration of TBM in combinationwith aromatic-cationic peptides will have synergistic effects in thisregard compared to that observed with either aromatic-cationic peptidesor TBMs alone.

These results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology are useful inthe treatment of vincristine-induced hyperalgesia, and drug-inducedhyperalgesia generally. The results will show that the TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates of the presenttechnology are useful in the treatment of drug-induced peripheralneuropathy or hyperalgesia.

Example 69—Use of Compositions of the Present Technology in thePrevention of Hyperalgesia in Humans

This Example will demonstrate use of the methods and compositions of thepresent technology in the prevention of hyperalgesia.

Subjects at risk for developing hyperalgesia will be recruited as theypresent in clinic for the treatment of conditions associated with thedevelopment of peripheral neuropathy or hyperalgesia. Independentstudies will address neuropathy and hyperalgesia resulting from, causedby, or otherwise associated with genetic disorders, metabolic/endocrinecomplications, inflammatory diseases, vitamin deficiencies, malignantdiseases, and toxicity, such as alcohol, organic metal, heavy metal,radiation, and drug toxicity. Subjects will be selected such that theyare at risk for developing a single type of neuropathy or hyperalgesia,having no risk factors outside the scope of the study in which thesubject is enrolled, and as yet not having symptoms associated withneuropathy or hyperalgesia. Subjects will be screened for theirwillingness to enroll in the study, and informed consent will beobtained. Healthy subjects will also be recruited for collection ofcomparison data.

After a focused interview about the medical history, baselinemeasurements of touch and sharpness detection thresholds, groovedpegboard tests, and thermal detection thresholds will be determinedaccording to the methods described above, with statistical analysis asdescribed above.

Following initial assessment of the above criteria, subjects will bedivided into the following groups:

a) Healthy controls

b) No treatment

c) Vehicle-only placebo, administered s.c., once daily

d) peptide conjugate, 10 mg/kg, administered s.c., once daily for 14days

e) aromatic-cationic peptide (equivalent molar doses ofaromatic-cationic peptide based on concentration of thearomatic-cationic peptide administered in the 10 mg/kg peptide conjugategroup), administered s.c., once daily for 14 days

f) TBM (equivalent molar doses of TBM based on concentration of the TBMadministered in the 10 mg/kg peptide conjugate group), administereds.c., once daily for 14 days

g) TBMs in combination with aromatic-cationic peptides (e.g., anequivalent molar dose of TBM based on the concentration of TBMadministered in the 10 mg/kg peptide conjugate treatment group and anequivalent molar dose of aromatic-cationic peptide based on theconcentration of aromatic-cationic peptide administered in the 10 mg/kgpeptide conjugate treatment group), administered s.c., once daily for 14days

Subjects will be evaluated weekly during the trial for sharpnessdetection thresholds, grooved pegboard tests, and thermal detectionthresholds. The trial will continue for a period of 28 days, or untilthe no-treatment and placebo control groups display hyperalgesiaaccording to the above criteria, at which point subjects will undergo afinal assessment.

Results—

It is expected that at-risk subjects that are treated with the peptideconjugate, aromatic-cationic peptide or TBM (with or withoutaromatic-cationic peptides) will show attenuated development ofneuropathy or hyperalgesia compared to untreated and placebo controls.It is anticipated that administration of peptide conjugates of thepresent technology will have synergistic effects in this regard comparedto that observed with either aromatic-cationic peptides or TBMs (aloneor in combination with aromatic-cationic peptides). It is anticipatedthat administration of TBM in combination with aromatic-cationicpeptides will have synergistic effects in this regard compared to thatobserved with either aromatic-cationic peptides or TBMs alone.

These results will show that the TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology are useful inthe prevention of neuropathy and hyperalgesia generally.

Example 70: Effects of Compositions of the Present Technology on HeartMitochondrial Cardiolipin in a Dog Model of Heart Failure

This Example demonstrates the effect of peptide conjugates on levels ofheart mitochondrial cardiolipin in dogs with coronarymicroembolization-induced heart failure. In particular, the effects ofD-Arg-2′6′-Dmt-Lys-Phe-NH₂-TBM conjugate on levels of the18:2-18:2-18:2-18:2 cardiolipin species are evaluated.

Heart failure is induced in dogs via multiple sequential intracoronarymicroembolizations as described in Sabbah, et al., Am J Physiol. (1991)260:H1379-84, herein incorporated by reference in its entirety. Group Idogs are subsequently treated with a daily dose of 0.25 mg/kg/day of thepeptide conjugate; Group II dogs are treated with only TBM at anequivalent molar dose of a daily dose of the TBM in the 0.25 mg/kg/daydose of the peptide conjugate; Group III dogs are treated withD-Arg-2′6′-Dmt-Lys-Phe-NH₂ at an equivalent molar dose of theD-Arg-2′6′-Dmt-Lys-Phe-NH₂ in the daily dose of 0.25 mg/kg/day of thepeptide conjugate; Group IV dogs are treated with TBMs in combinationwith D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (e.g., an equivalent molar dose of TBMbased on the concentration of TBM administered in the 0.25 mg/kg/daydose of the peptide conjugate treatment group and an equivalent molardose of D-Arg-2′6′-Dmt-Lys-Phe-NH₂ based on the concentration ofD-Arg-2′6′-Dmt-Lys-Phe-NH₂ administered in the 0.25 mg/kg/day dose ofthe peptide conjugate treatment group). Group V dogs are treated withdrug vehicle and serve as controls. Treatment with the various agents ofGroups I, II, III, and IV are started upon induction of heart failure(HF), defined as left ventricular ejection fraction of approximately30%. Doses are administered intravenously. At the end of the treatmentphase (12 weeks), dogs in the control and treatment groups aresacrificed and a sample of heart muscle from the left ventricle isremoved, washed with saline, and immediately frozen and stored at −80°C. For cardiolipin analysis, lipids are extracted from the heart tissuesample with a chloroform/methanol solution (Bligh Dyer extraction).Individual lipid extracts are reconstituted with chloroform:methanol(1:1), flushed with N2, and then stored at −20° C. before analysis viaelectrospray ionization mass spectroscopy using a triple-quadrupole massspectrometer equipped with an automated nanospray apparatus. Enhancedmultidimensional mass spectrometry-based shotgun lipidomics forcardiolipin is performed as described by Han, et al., “Shotgunlipidomics of cardiolipin molecular species in lipid extracts ofbiological samples,” J Lipid Res 47(4)864-879 (2006).

It is anticipated that the levels of 18:2 cardiolipin species will besignificantly reduced in untreated heart failure dogs (Heart Failure,Control) as compared to cardiac tissue from normal subjects (Normal). Itis further anticipated that subjects treated withD-Arg-2′6′-Dmt-Lys-Phe-NH₂-TBM conjugates, TBM (alone or in combinationwith D-Arg-2′6′-Dmt-Lys-Phe-NH₂), or D-Arg-2′6′-Dmt-Lys-Phe-NH₂ willhave 18:2 cardiolipin levels that are similar to normal subjects, andgreater than the heart failure control subjects. It is anticipated thatadministration of peptide conjugates of the present technology will havesynergistic effects in this regard (e.g., D-Arg-2′6′-Dmt-Lys-Phe-NH₂-TBMconjugates are more therapeutically effective at normalizing cardiolipinlevels compared to treatment with either D-Arg-2′6′-Dmt-Lys-Phe-NH₂ orTBM (alone or in combination with D-Arg-2′6′-Dmt-Lys-Phe-NH₂)). It isanticipated that administration of TBM in combination withD-Arg-2′6′-Dmt-Lys-Phe-NH₂ will have synergistic effects in this regardcompared to that observed with either D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or TBMsalone.

The results will show that TBMs (with or without aromatic-cationicpeptides) or peptide conjugates of the present technology are useful inthe prevention and treatment of diseases and conditions associated withaberrant cardiolipin levels. These results show that TBMs (with orwithout aromatic-cationic peptides) or peptide conjugates of the presenttechnology are useful in methods comprising administration of thepeptide conjugates to subjects in need of normalization of cardiolipinlevels and remodeling.

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presenttechnology is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this present technology is notlimited to particular methods, reagents, compounds compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

What is claimed is:
 1. A method for delivering an aromatic-cationicpeptide and/or a therapeutic biological molecule to a cell, the methodcomprising contacting the cell with a peptide conjugate, wherein thepeptide conjugate comprises the therapeutic biological moleculeconjugated to an aromatic-cationic peptide, wherein thearomatic-cationic peptide is selected from the group consisting of:2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, andD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, and wherein the therapeutic biologicalmolecule is frataxin.
 2. A method according claim 1, wherein thetherapeutic biological molecule is conjugated to the aromatic-cationicpeptide by a linker.
 3. A method according claim 1, wherein thetherapeutic biological molecule and aromatic-cationic peptide arechemically bonded.
 4. A method according claim 1, wherein thetherapeutic biological molecule and aromatic-cationic peptide arephysically bonded.
 5. A method according claim 2, wherein thearomatic-cationic peptide and the therapeutic biological molecule arelinked using a labile linkage that is hydrolyzed in vivo to uncouple thearomatic-cationic peptide and the therapeutic biological molecule.
 6. Amethod according claim 5, wherein the labile linkage comprises an esterlinkage.
 7. A peptide conjugate comprising a therapeutic biologicalmolecule conjugated to an aromatic-cationic peptide, wherein thearomatic-cationic peptide is Phe-D-Arg-Phe-Lys-NH₂ orD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, and wherein the therapeutic biologicalmolecule is frataxin.
 8. A peptide conjugate according to claim 7,wherein the therapeutic biological molecule is conjugated to thearomatic-cationic peptide by a linker.
 9. A peptide conjugate accordingto claim 7, wherein the therapeutic biological molecule andaromatic-cationic peptide are chemically bonded.
 10. A peptide conjugateaccording to claim 7, wherein the therapeutic biological molecule andaromatic-cationic peptide are physically bonded.
 11. A peptide conjugateaccording to claim 7, wherein the aromatic-cationic peptide and thetherapeutic biological molecule are linked using a labile linkage thatis hydrolyzed in vivo to uncouple the aromatic-cationic peptide and thetherapeutic biological molecule.
 12. A peptide conjugate according toclaim 11, wherein the labile linkage comprises an ester linkage.